Methods And Compositions For Detecting Fungi And Mycotoxins

ABSTRACT

The invention relates to a method of identifying a specific fungal species in patient tissue or body fluid. The method comprises the steps of extracting and recovering DNA of the fungal species from the patient tissue or body fluid, amplifying the DNA, hybridizing a probe to the DNA to specifically identify the fungal species, and specifically identifying the fungal species. The invention also relates to a method of identifying a mycotoxin in patient tissue or body fluid. The method comprises the steps of extracting and recovering the mycotoxin from the patient tissue or body fluid, contacting the mycotoxin with an antibody directed against the mycotoxin, and identifying the myocotoxin. Both of these methods can be used to determine if a patient is at risk for or has developed a disease state related to a fungal infection, and to develop an effective treatment regimen for the patient.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C §119(e) to U.S.Provisional Application Ser. Nos. 60/787,754, filed on Apr. 1, 2006, and60/790,974, filed on Apr. 11, 2006.

FIELD OF THE INVENTION

This invention relates to methods and compositions for detecting oridentifying fungi or for detecting or identifying mycotoxins. Moreparticularly, the invention relates to methods and compositions fordetecting or identifying fungi and for detecting or identifyingmycotoxins in the tissues or body fluid samples of patients.

BACKGROUND AND SUMMARY

Molds (i.e., toxigenic and other septate molds) are ubiquitous in theenvironment. Mold is the common name for various types of fungi. Moldsare usually found in moist, warm environments. Because molds grow in wetor moist indoor environments, people are exposed to molds or theirbyproducts through either direct contact, or through the air, if moldsor mold byproducts are aerosolized. Exposure to molds can cause a numberof adverse effects including allergic reactions, asthma attacks, andinfections, particularly in individuals with immune system deficiencies.

Adverse effects from molds may occur when individuals are exposed tolarge doses of chemicals, known as mycotoxins, which are fungalmetabolites (Samson et al., 1985; Burge, 1990; Flannigan et al., 1991).Mycotoxins have toxic effects ranging from severe irritations, such asallergic reactions and asthma, to immuno-suppression and cancer. Mostmycotoxins are cytotoxic and exert their effects by interfering withvital cellular processes such as protein, RNA, and DNA synthesis. As aresult, mycotoxins may be damaging to the skin, the lungs, the gut, andthe like. The combined outcome may increase the susceptibility of theexposed individual to infectious diseases and, possibly, to cancer.Almost all of the studies to date focus on disease induced by mycotoxinsingested in contaminated food (Baxter et al., 1981), but mycotoxins aresecondary metabolites of fungal spores and can enter the body throughthe respiratory tract.

In heavily contaminated environments, neurotoxic symptoms related toairborne mycotoxin exposure have been reported (Croft et al., 1986).Skin is another potential route of exposure to the mycotoxins of severalfungi which have caused cases of severe dermatosis (Vennewald andWollina, 2005). These same molds may cause invasive mold infection amongpatients with diseases which render the patient immuno-suppressed suchas leukemia, lymphoma, and many cancers (Kontoyiannis, DP et al, 2005).The mold infections in such patients are often fatal with a documentedfatally rate of 92% (Paterson and Singh, 1999).

There are no current methods that have been developed for determiningthe presence of mycotoxins in patient tissues or body fluids. There are,however, methods available to the environmental areas and to the foodindustry to determine levels of mycotoxins, such as tricothecenes,Aflatoxins B1, B2, D1, and D2, and Ochratoxin A (e.g., Envirologix andVICAM kits) in environmental samples and foods.

A definitive and early diagnosis of a fungal infection is crucial forpatient treatment and management. A diagnosis of a fungal infection isoften rendered late in the disease process, often even as late asautopsy (Kontoyiannis et al, 2000; Vogeser et al., 1997). The reasonsfor the late diagnosis of fungal infections include the lack of goodclinical specimens, the difficultly in differentiating invasive moldinfections from other types of infections, the lack of identification ofmolds with special stains in pathological specimens (i.e., these assayshave a high error rate, a low sensitivity, and low specificity), thelack of an ability to obtain an antibody-based diagnosis inimmuno-compromised patients, and the lack of assays to determine thepresence of mycotoxins or fungal DNA in the tissue or fluids of thosepatients.

Thus, a reliable, sensitive, specific, and rapid method for molddetection in patient body fluids and tissues is needed. Applicant'spresent invention is based on the idea that if mycotoxins can beidentified in patient tissue or body fluids, the identification ofmycotoxins may serve as a potential diagnostic method 1.) to identifypatients at risk for developing disease states related to moldinfections, or 2.) to rapidly determine the cause of diseases related tomold infections so that effective treatment regimens can be developedfor patients exposed to molds and experiencing symptoms resulting frommold infection. Applicant's present invention is also based on thedevelopment of a reliable, sensitive, specific, and rapid method fordetecting fungal DNA in patient body fluids and tissues.

The present invention provides methods for detecting and identifying, inpatient tissue and body fluid specimens, 1.) mycotoxins produced byfungi, and 2.) fungal DNA from fungal spores. The present inventionovercomes the deficiencies in the art by providing reliable, sensitive,and specific diagnostic tests for the presence of fungi and fungaltoxins in patient tissue and body fluids. Applicant has developedmycotoxin and fungal DNA extraction procedures and has supplementedthose methods by developing detection methods. The detection methodsemploy antibody-based identification for mycotoxins and, for fungal DNA,use amplification of DNA with primers that specifically and selectivelyamplify fungal DNA isolated from patient tissues and body fluids.

In one illustrative embodiment, a method is provided of identifying aspecific fungal species in patient tissue or body fluid. The methodcomprises the steps of extracting and recovering DNA of the fungalspecies from the patient tissue or body fluid, amplifying the DNA,hybridizing a probe to the DNA to specifically identify the fungalspecies, and specifically identifying the fungal species.

In another embodiment, a method is provided of identifying a mycotoxinin patient tissue or body fluid. The method comprises the steps ofextracting and recovering the mycotoxin from the patient tissue or bodyfluid, contacting the mycotoxin with an antibody directed against themycotoxin, and identifying the myocotoxin. In another illustrativeembodiment the method can further comprise the step of quantifying themycotoxin. In illustrative embodiments, the body fluid can be selectedfrom the group consisting of urine, nasal secretions, nasal washes,bronchial lavages, bronchial washes, spinal fluid, sputum, gastricsecretions, seminal fluid, other reproductive tract secretions, lymphfluid, whole blood, serum, and plasma. In other illustrativeembodiments, the mycotoxin can be selected from the group consisting oftricothecenes, Aflatoxin B1, Aflatoxin B2, Aflatoxin D1, Aflatoxin D2,and Ochratoxin A. In yet other illustrative embodiments, the tissue canbe derived from a patient tissue biopsy and can be in a 10% formalinsolution or in a paraffin block. In another embodiment, the antibody isbound to a bead dyed with a fluorochrome.

In yet another embodiment, a method is provided of determining if apatient is at risk for or has developed a disease state related to afungal infection. The method comprises the steps of extracting andrecovering a mycotoxin from a tissue or body fluid of the patient,contacting the mycotoxin with an antibody directed against the toxin,identifying the mycotoxin, and determining if the patient is at risk foror has developed the disease state related to the fungal infection.

In still another embodiment, a method is provided of determining if apatient is at risk for or has developed a disease state related to afungal infection. The method comprises the steps of extracting andrecovering DNA of a specific fungal species from a tissue or body fluidof the patient, amplifying the DNA, hybridizing a probe to the DNA tospecifically identify the fungal species, and specifically identifyingthe fungal species.

In another embodiment, a kit is provided. The kit can comprise any oneof the probes described herein and/or any one of the primer setsdescribed herein. The kit can also comprise components for theextraction and recovery of DNA and components for DNA amplification andinstructions for use of the kit.

In yet another embodiment, a kit is provided. The kit comprisescomponents for the extraction and recovery of a mycotoxin from bodyfluid or tissue of a patient. In other embodiments, the kit can furthercomprise components for identification of the mycotoxin and instructionsfor use of the kit.

In still another embodiment, a kit is provided. The kit comprises apurified nucleic acid with a sequence selected from the group consistingof SEQ ID NO: 1 to SEQ ID NO: 89 or with a complement of a sequenceselected from the group consisting of of SEQ ID NO: 1 to SEQ ID NO: 89.

In another illustrative embodiment, a purified nucleic acid is provided.The nucleic acid comprises a sequence of SEQ ID NO: 1 to SEQ ID NO: 89or a complement of a sequence of SEQ ID NO: 1 to SEQ ID NO: 89. Inanother illustrative embodiment, a nucleic acid is provided thathybridizes under highly stringent conditions to a sequence comprising asequence of SEQ ID NO: 1 to SEQ ID NO: 89 or that hybridizes underhighly stringent conditions to a complement of a sequence of SEQ ID NO:1 to SEQ ID NO: 89.

In yet another embodiment, a method of detecting an antibody to amycotoxin in a patient tissue extract or body fluid is provided. Themethod comprises the steps of contacting the patient tissue extract orbody fluid with a mycotoxin or a mycotoxin antigen coupled to a beadwherein the bead is dyed with a fluorochrome, and detecting theantibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Tricothecenes values for urine tests.

FIG. 2 shows Tricothecenes values for nasal secretions.

FIG. 3 shows fungal DNA coupling efficiency to microspheres.

FIG. 4 shows mycotoxin coupling efficiency to microspheres.

DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS

The present invention relates to methods and compositions foridentifying or detecting the presence of molds (i.e., fungi) in patienttissue and body fluids. The identification and detection methods arebased on 1.) amplification of fungal DNA using a PCR-based method and2.) detection and quantification of mycotoxins in patient body fluidsand tissues. The methods and compositions (e.g., primers and probes) foramplification of fungal DNA are highly specific and sensitive and avoidco-amplification of or do not co-amplify non-specific human or animalnucleic acids.

The methods and compositions for detection and quantification ofmycotoxins are also very specific and sensitive. These methods andcompositions utilize antibody-based identification of mycotoxins. Inillustrative embodiments, Enzyme Linked Immunosorbant Assay (ELISA), oraffinity chromatography can be used to detect mycotoxins produced bytoxic molds. Illustratively, the mycotoxins can be aflatoxins,ochratoxins, or tricothecenes (e.g., Verrucarins A, B and J, Roridin A,E, H, and L-2, Satratoxins F, G, and H, Verrucarol, isosatratoxin F, G,and H, and T-2). Illustrative of antibody-based assays that can be usedto identify mycotoxins are the Tricothecene kit (Envirologix, Inc.,Portland, Me.), the AflaTest® (VICAM, Inc.), the OchraTest™ (VICAMInc.), and Luminex®-based assays.

Aflatoxins are toxin metabolites produced by a variety of molds such asAspergillus flavus and Aspergillus parasiticus. Aflatoxins arecarcinogenic and can be present in grains, nuts, cottonseed and othercommodities associated with human food or animal feeds. Crops may becontaminated by one or more of the four sub-types of aflatoxins. B1, B2,G1 and G2. Aflatoxin B 1 is the most toxic and frequently detectedaflatoxin. Aflatoxins have been implicated in human health disordersincluding hepatocellular carcinoma, Reye's syndrome, and chronichepatitis.

Ochratoxin A is the most important and most commonly occurring of astructurally related group of compounds called ochratoxins. Ochratoxin Ais produced by some species of Aspergillus, such as A. ochraceus, and byPenicillium verrucosum. Ochratoxin A is a potent toxin affecting mainlythe kidneys, in which it can cause both acute and chronic lesions. Anephrotoxic effect has been demonstrated in all mammalian species, andboth teratogenic and reproductive effects have been demonstrated.Ochratoxin A is known to affect the immune system in a number ofmammalian species.

Some tricothecenes are macrocyclic mycotoxins. There are over onehundred trichothecenes that cause irritation and immunosuppressiveeffects (Ueno, Y. 1983 and Tuomi, et al. 2000). Most tricothecenes wereoriginally isolated from species of Fusarium, but they also may beproduced by other fungi, such as species of Stachybotrys, Trichothecium,and others (Kurata and Ueno, 1983 and Ueno, Y. 1983). Stachybotryschartarum is known to produce a number of potent mycotoxins in thisfamily including Verrucarins B and J, Roridin E, Satratoxins F, G, andH, and isosatratoxin F, G, and H (Hinkley, et al. 2001 and Jarvis, etal, 1998). These mycotoxins are also known to be potent inhibitors ofprotein synthesis in eukaryotes.

In various illustrative embodiments, body fluids that can be tested forthe presence of fungal DNA or mycotoxins, include, but are not limitedto, urine, nasal secretions, nasal washes, inner ear fluids, bronchiallavages, bronchial washes, alveolar lavages, spinal fluid, bone marrowaspirates, sputum, pleural fluids, synovial fluids, pericardial fluids,peritoneal fluids, saliva, tears, gastric secretions, stool,reproductive tract secretions, such as seminal fluid, lymph fluid, andwhole blood, serum, or plasma. These samples can be prepared for testingas described herein. In various embodiments, tissue samples can includetissue biopsies of hospital patients or out-patients and autopsyspecimens. As used herein, the term “tissue” includes, but is notlimited to, biopsies, autopsy specimens, cell extracts, tissue sections,aspirates, tissue swabs, and fine needle aspirates.

In accordance with the invention the word “patient” means a human or ananimal, such as a domestic animal (e.g., a dog or a cat). Accordingly,the methods and compositions disclosed herein can be used for both humanclinical medicine and veterinary applications. Thus, the patientafflicted with a disease state related to a fungal infection can be ahuman, or in the case of veterinary applications, can be a laboratory,agricultural, domestic or wild animal. The present invention can beapplied to patients including, but not limited to, humans, laboratoryanimals such rodents (e.g., mice, rats, hamsters, etc.), rabbits,monkeys, chimpanzees, domestic animals such as dogs, cats, and rabbits,agricultural animals such as cows, horses, pigs, sheep, goats, chickens,and wild animals in captivity such as bears, pandas, lions, tigers,leopards, elephants, zebras, giraffes, gorillas, dolphins, and whales.

The methods and compositions described herein can be used to detect oridentify microbial DNA (e.g., fungal DNA) or microbial toxins (e.g.,mycotoxins) in microbes selected from the group consisting of Absidiacoerulea, Absidia glauca, Absidia corymbifera, Acremonium strictum,Alternaria alternata, Apophysomyces elegans, Saksena vasiformis,Aspergillus flavus, Aspergillus oryzae, Aspergillus fumigatus,Neosartoryta fischeri, Aspergillus niger, Aspergillus foetidus,Aspergillus phoenicus, Aspergillus nomius, Aspergillus ochraceus,Aspergillus ostianus, Aspergillus auricomus, Aspergillus parasiticus,Aspergillus sojae, Aspergillus restrictus, Aspergillus caesillus,Aspergillus conicus, Aspergillus sydowii, Aspergillus tamarii,Aspergillus terreus, Aspergillus ustus, Aspergillus versicolor,Aspergillus ustus, Aspergillus versicolor, Chaetomium globosum,Cladosporium cladosporioides, Cladosporium herbarum, Cladosporiumsphaerospermum, Conidiobolus coronatus, Conidiobolus incongruus,Cunninghamella elegans, Emericella nidulans, Emericella rugulosa,Emericilla quadrilineata, Apicoccum nigrum, Eurotium amstelodami,Eurotium chevalieri, Eurotium herbariorum, Eurotium rubrum, Eurotiumrepens, Geotrichum candidum, Geotrichum klebahnii, Memnoniella echinata,Mortierella polycephala, Mortierella wolfii, Mucor mucedo, Mucoramphibiorum, Mucor circinelloides, Mucor heimalis, Mucor indicus, Mucorracemosus, Mucor ramosissimus, Rhizopus azygosporous, Rhizopushomothalicus, Rhizopus microsporus, Rhizopus oligosporus, Rhizopusoryzae, Myrothecium verrucaria, Myrothecium roridum, Paecilomyceslilacinus, Paecilomyces variotii, Penicillium freii, Penicilliumverrucosum, Penicillium hirsutum, Penicillium alberechii, Penicillumaurantiogriseum, Penicillium polonicum, Penicillium viridicatum,Penicillium hirsutum, Penicillium brevicompactum, Penicilliumchrysogenum, Penicillium griseofulvum, Penicillium glandicola,Penicillium coprophilum, Eupenicillium crustaceum, Eupenicilliumegyptiacum, Penicillium crustosum, Penicillium citrinum, Penicilliumsartoryi, Penicillium westlingi, Penicillium corylophilum, Penicilliumdecumbens, Penicillium echinulatum, Penicillium solitum, Penicilliumcamembertii, Penicillium commune, Penicillium echinulatum, Penicilliumsclerotigenum, Penicillium italicum, Penicillium expansum, Penicilliumfellutanum, Penicillium charlesii, Penicillium janthinellum, Penicilliumraperi, Penicillium madriti, Penicillium gladioli, Penicillium oxalicum,Penicillium roquefortii, Penicillium simplicissimum, Penicilliumochrochloron, Penicillium spinulosum, Penicillium glabrum, Penicillumthomii, Penicillium pupurescens, Eupenicillium lapidosum, Rhizomucormiehei, Rhizomucor pusillus, Rhizomucor variabilis, Rhizopus stolonifer,Scopulariopsis asperula, Scopulariopsis brevicaulis, Scopulariopsisfusca, Scopulariopsis brumptii, Scopulariopsis chartarum, Scopulariopsissphaerospora, Trichoderma asperellum, Trichoderma hamatum, Trichodermaviride, Trichoderma harzianum, Trichoderma longibrachiatum, Trichodermacitroviride, Trichoderma atroviride, Trichoderma koningii, Ulocladiumatrum, Ulocladium chartarum, Ulocladium botrytis, Wallemia sebi,Stachybotrys chartarum, and the like.

In embodiments where the microbe is a fungal species, the microbe istypically selected from the group consisting of S. chartarum, S.prolificans, A. versicolor, A. vesicularis, A. niger, P. chrysogenum, P.verrucosum, G. candidum, A. flavus, A. fumigatus, A. nidulans, A.ochraceus, A. paraciticus, A. sydowii, A. ustus, F. solani, F.chlamydosporum, P. aurantiogriseum, P. citrinum, P. corylophilum, P.crustosum, P. expansum, P. fellutanum, P. roquefortii, P.simplicissimum, S. echinata, and E. amstelodami. In one embodiment, themolds (i.e., fungi) can be black, toxigenic molds.

In one illustrative embodiment, a method is provided of identifying aspecific fungal species in patient tissue or body fluid. The methodcomprises the steps of extracting and recovering DNA of the fungalspecies from the patient tissue or body fluid, amplifying the DNA,hybridizing a probe to the DNA to specifically identify the fungalspecies, and specifically identifying the fungal species.

In some embodiments, real-time PCR-based methods can be used to amplifythe fungal DNA and to detect and identify fungal DNA by hybridization ofthe probe to the fungal DNA. PCR is described in U.S. Pat. Nos.4,683,202 and 4,800,159, incorporated herein by reference, and methodsfor PCR are well-known in the art. Real-time PCR combines amplificationand simultaneous probe hybridization to achieve sensitive and specificdetection of infectious molds (i.e., fungi) in real-time therebyproviding instant detection of molds. In this embodiment, the time todetect or identify the fungus and to obtain a diagnosis is greatlyreduced. Real-time PCR is conducted according to methods well-known inthe art. Exemplary probes and primers and their target DNAs, that can beused in accordance with the invention are shown below. “Primer F” refersto a forward primer and “Primer R” refers to a reverse primer which arewell-known terms in the art.

Target 1 - S. chartarum (SEQ ID NO: 1)Probe 1 char: 5′-ttgcttcggcgggaacgccccg  (SEQ ID NO: 2)Primer F2: 5′-gcggagggatcattaccgag (SEQ ID NO: 3)Primer R2: 5′-atcgatgccagagccaagag Target 2 - A. versicolor(SEQ ID NO: 4) Probe 2 vers: 5′-cggggagccactcgggggc (SEQ ID NO: 5)Primer F1: 5′-cgtaggtgaacctgcggaag (SEQ ID NO: 6)Primer R1: 5′-atcgatgccggaaccaagag Target 3 - A. niger (SEQ ID NO: 7)Probe 3 niger: 5′-tgtctattgtacctgttgcttc (SEQ ID NO: 8)Primer F14: 5′-cgtaggtgaacctgcggaag (SEQ ID NO: 9)Primer R1: 5′-atcgatgccggaaccaagag Target 4 - P. chrysogenum(SEQ ID NO: 10) Probe 4 chry: 5′-ctctgtctgaagattgtagtctgagt(SEQ ID NO: 11) Primer F1: 5′-cgtaggtgaacctgcggaag (SEQ ID NO: 12)Primer R1: 5′-atcgatgccggaaccaagag Target 5 - P. verrucosum(SEQ ID NO: 13) Probe 5 verru: 5′-cccgcctttgctggccgcc (SEQ ID NO: 14)Primer F1: 5′-cgtaggtgaacctgcggaag (SEQ ID NO: 15)Primer R1: 5′-atcgatgccggaaccaagag Target 6 - G. candidum(SEQ ID NO: 16) For Geo F1H: 5′-ggatctcttggttctcgtatc (SEQ ID NO: 17)Rev Geo R1H: 5′-cttgatctgaggttgaatagtg (SEQ ID NO: 18)Probe 6 geo: 5″-aacgcacattgcactttggggtatc Target 7 - A. flavus(SEQ ID NO: 19) Probe 7 flav: 5′-cccgccattcatggccgccggg (SEQ ID NO: 20)Primer F1: 5′-cgtaggtgaacctgcggaag (SEQ ID NO: 21)Primer R1: 5′-atcgatgccggaaccaagag Target 8 - A. fumigatus(SEQ ID NO: 22) Probe 8 fumi: 5′-aaagtatgcagtctgagttgattatc(SEQ ID NO: 23) Primer F1: 5′-cgtaggtgaacctgcggaag (SEQ ID NO: 24)Primer R1: 5′-atcgatgccggaaccaagag Target 9 - A. nidulans(SEQ ID NO: 25) Probe 9 nid: 5′-cccagggggcgagccgccgg (SEQ ID NO: 26)Primer F1: 5′-cgtaggtgaacctgcggaag (SEQ ID NO: 27)Primer R1: 5′-atcgatgccggaaccaagag Target 10 - A. ochraceus(SEQ ID NO: 28) Probe 10 ochr: 5′-acaccaacgtgaacactgtctgaag(SEQ ID NO: 29) Primer F1: 5′-cgtaggtgaacctgcggaag (SEQ ID NO: 30)Primer R1: 5′-atcgatgccggaaccaagag Target 11 - A. paraciticus(SEQ ID NO: 31) Probe 11 para: 5′-cgggcccgccgtcatggccg (SEQ ID NO: 32)Primer F1: 5′-cgtaggtgaacctgcggaag (SEQ ID NO: 33)Primer R1: 5′-atcgatgccggaaccaagag Target 12 - A. sydowii(SEQ ID NO: 34) Probe 12 syd: 5′-ccctcgggggcgagccgccg (SEQ ID NO: 35)Primer F1: 5′-cgtaggtgaacctgcggaag (SEQ ID NO: 36)Primer R1: 5′-atcgatgccggaaccaagag Target 13 - A. ustus (SEQ ID NO: 37)Probe 13 ust: 5′-ccacaccgaacctcttgttatagc (SEQ ID NO: 38)Primer F1: 5′-cgtaggtgaacctgcggaag (SEQ ID NO: 39)Primer R1: 5′-atcgatgccggaaccaagag Target 14 - F. solani (SEQ ID NO: 40)Probe14salani: 5′-cgggaatagacggccccgtgaaac (SEQ ID NO: 41)Primer F2: 5′-gcggagggatcattaccgag (SEQ ID NO: 42)Primer R2: 5′-atcgatgccagagccaagag Target 15 - P. aurantiogriseum(SEQ ID NO: 43) Probe 15 auran: 5′-cccgcctttactggccgccgg (SEQ ID NO: 44)Primer F1: 5′-cgtaggtgaacctgcggaag (SEQ ID NO: 45)Primer R1: 5′-atcgatgccggaaccaagag Target 16 - P. citrinum(SEQ ID NO: 46) Probe 16 citr: 5′-tgttgcctcggcgggccccgc (SEQ ID NO: 47)Primer F4: 5′-ggaaggatcattaccgagtg (SEQ ID NO: 48)Primer R1: 5′-atcgatgccggaaccaagag Target 17 - P. corylophilum(SEQ ID NO: 49) Probe 17 corylo: 5′-ttattgtaccttgttgcttcggcgg(SEQ ID NO: 50) Primer F1: 5′-cgtaggtgaacctgcggaag (SEQ ID NO: 51)Primer R1: 5′-atcgatgccggaaccaagag Target 18 - P. crustosum(SEQ ID NO: 52) Probe 18 crust: 5′-cgatctccgggggacgggcc (SEQ ID NO: 53)Primer F7: 5′-ctgtccgagcgtcattgctg (SEQ ID NO: 54)Primer R5: 5′-cgaggaccggacgcggtg Target 19 - P. expansum (SEQ ID NO: 55)Probe19expan: 5′-agacacccccgaactctgcctgaa (SEQ ID NO: 56)Primer F1: 5′-cgtaggtgaacctgcggaag (SEQ ID NO: 57)Primer R1: 5′-atcgatgccggaaccaagag Target 20 - P. fellutanum(SEQ ID NO: 58) Probe 20 fell: 5′-cccgcctgccaggccgccg (SEQ ID NO: 59)Primer F1: 5′-cgtaggtgaacctgcggaag (SEQ ID NO: 60)Primer R1: 5′-atcgatgccggaaccaagag Target 21 - P. roquefortii(SEQ ID NO: 61) Probe 21 roque: 5′-cacccgtgtttatttaccttattgc(SEQ ID NO: 62) Primer F1: 5′-cgtaggtgaacctgcggaag (SEQ ID NO: 63)Primer R1: 5′-atcgatgccggaaccaagag Target 22 - P. simplicissimum(SEQ ID NO: 64) Probe 22 simpl: 5′-cacccgtgtttatcgtaccttgttg(SEQ ID NO: 65) Primer F1: 5′-cgtaggtgaacctgcggaag (SEQ ID NO: 66)Primer R1: 5′-atcgatgccggaaccaagag Target 23 - S. echinata(SEQ ID NO: 67) Probe 23 echin: 5′-ttgcttcggcgggagagccccg(SEQ ID NO: 68) Primer F2: 5′-gcggagggatcattaccgag (SEQ ID NO: 69)Primer R2: 5′-atcgatgccagagccaagag Target 24 - E. amstelodami(SEQ ID NO: 70) Probe 24 amst: 5′-tgtctatctgtaccctgttgcttcg(SEQ ID NO: 71) Primer F1: 5′-cgtaggtgaacctgcggaag (SEQ ID NO: 72)Primer R1: 5′-atcgatgccggaaccaagag Fungal Universal Group 1(SEQ ID NO: 73) UP1: 5′-cctcggatcaggtagggatac (SEQ ID NO: 74)UF1: 5′-atgcctgtccgagcgtcatt (SEQ ID NO: 75)UR1: 5′-ttcctccgcttattgatatg Fungal Universal Group 2 (SEQ ID NO: 76)Up2: 5′-acggatctcttggctctggcatc (SEQ ID NO: 77)F2: 5′-gcggagggatcattaccgag (SEQ ID NO: 78)UR2: 5′-ttcactgaattctgcaattcac

Alternative illustrative embodiments for the Target 3 probe and primerF1 are 5′-cctctgccccccgggcccgtg (SEQ ID NO: 79) and5′-ggaaggatcattaccgagtg (SEQ ID NO: 80), respectively. An alternativeillustrative embodiment for the Target 9 probe is5′-ggagccccccagggggcgag (SEQ ED NO: 81). An alternative illustrativeembodiment for the Target 12 probe is 5′-cggggaacccectegggggc (SEQ IDNO: 82). An alternative illustrative embodiment for the Target 13 probeis 5′-tgcgctccccccgggggcag (SEQ ID NO: 83). Alternative illustrativeembodiments for the Target 18 probe, primer F7, and primer R5 are5′-ggccccgtcceccgatctccg (SEQ ID NO: 84), 5′-agtgaatcatcgagtctttgaac(SEQ ID NO: 85), and 5′-acctgatccgaggtcaacctg (SEQ ID NO: 86),respectively. An alternative illustrative embodiment for the Target 20probe is 5′-cgggcccgcctgccaggccg (SEQ ID NO: 87). An alternativeillustrative embodiment for the Target 21 probe is5′-ccggggggtttacacccccg (SEQ ID NO: 88). An alternative illustrativeembodiment for the Target 22 probe is 5′-coggggggcatetgecccegg (SEQ IDNO: 89).

In various embodiments, sample preparation (i.e., preparation of thetarget DNA) involves rupturing the cells (e.g., cells of the tissue orfungal spores in patient body fluid or tissue) and isolating the fungalDNA from the lysate. Techniques for rupturing cells and for isolation ofDNA are well-known in the art. For example, cells may be ruptured byusing a detergent or a solvent, such as phenol-chloroform. DNA may beseparated from the lysate by physical methods including, but not limitedto, centrifugation, pressure techniques, or by using a substance withaffinity for DNA, such as, for example, silica beads. After sufficientwashing, the isolated DNA may be suspended in either water or a buffer.In other embodiments, commercial kits are available, such as Quiagen™,Nuclisensm™, and Wizard™ (Promega), and Promegam™. Methods for isolatingDNA are described in Sambrook et al., “Molecular Cloning: A LaboratoryManual”, 3rd Edition, Cold Spring Harbor Laboratory Press, (2001),incorporated herein by reference.

In various embodiments described herein, the primers and probes used foramplification of the target DNA and for detection and identification offungal DNA are oligonucleotides from about ten to about one hundred,more typically from about ten to about thirty or about six to abouttwenty-five base pairs long, but any suitable sequence length can beused. In illustrative embodiments, the primers and probes may bedouble-stranded or single-stranded, but the primers and probes aretypically single-stranded. The primers and probes described herein arecapable of specific hybridization, under appropriate hybridizationconditions (e.g., appropriate buffer, ionic strength, temperature,formamide, and MgCl₂ concentrations), to a region of the target DNA. Theprimers and probes described herein are designed based on having amelting temperature within a certain range, and substantialcomplementarity to the target DNA. Methods for the design of primers andprobes are described in Sambrook et al., “Molecular Cloning: ALaboratory Manual”, 3rd Edition, Cold Spring Harbor Laboratory Press,(2001), incorporated herein by reference.

The primers and probes described herein for use in PCR can be modifiedby substitution, deletion, truncation, and/or can be fused with othernucleic acid molecules wherein the resulting primers and probeshybridize specifically to the intended targets and are useful in themethods described herein for amplification of the target DNAs.Derivatives can also be made such as phosphorothioate, phosphotriester,phosphoramidate, and methylphosphonate derivatives, that specificallybind to single-stranded DNA or RNA (Goodchild, et al., Proc. Natl. Acad.Sci. 83:4143-4146 (1986)).

The invention encompasses isolated or substantially purified nucleicacids. An “isolated” or “purified” nucleic acid molecule issubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. Preferably,an “isolated” or “purified” nucleic acid is free of sequences thatnaturally flank the nucleic acid in the genomic DNA of the organism fromwhich the nucleic acid is derived. For example, in various embodiments,the isolated or purified nucleic acid molecule can contain less thanabout 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotidesequences that naturally flank the nucleic acid molecule in genomic DNAof the cell from which the nucleic acid is derived.

Also within the scope of the invention are nucleic acids complementaryto the probes and primers described herein, and those that hybridize tothe nucleic acids described herein or those that hybridize to theircomplements under highly stringent conditions. In accordance with theinvention “highly stringent conditions” means hybridization at 65° C. in5×SSPE and 50% formamide, and washing at 65° C. in 0.5×SSPE. Conditionsfor low stringency and moderately stringent hybridization are describedin Sambrook et al., “Molecular Cloning: A Laboratory Manual”, 3rdEdition, Cold Spring Harbor Laboratory Press, (2001), incorporatedherein by reference. In some illustrative aspects, hybridization occursalong the full-length of the nucleic acid.

Also included are nucleic acid molecules having about 60%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, 96%, 97%, and 98%homology to the probes and primers described herein. Determination ofpercent identity or similarity between sequences can be done, forexample, by using the GAP program (Genetics Computer Group, software;now available via Accelrys on http://www.accelrys.com), and alignmentscan be done using, for example, the ClustalW algorithm (VNTI software,InforMax Inc.). A sequence database can be searched using the nucleicacid sequence of interest. Algorithms for database searching aretypically based on the BLAST software (Altschul et al., 1990). In someembodiments, the percent identity can be determined along thefull-length of the nucleic acid.

As used herein, the term “complementary” refers to the ability of purineand pyrimidine nucleotide sequences to associate through hydrogenbonding to form double-stranded nucleic acid molecules. Guanine andcytosine, adenine and thymine, and adenine and uracil are complementaryand can associate through hydrogen bonding resulting in the formation ofdouble-stranded nucleic acid molecules when two nucleic acid moleculeshave “complementary” sequences. The complementary sequences can be DNAor RNA sequences. The complementary DNA or RNA sequences are referred toas a “complement.”

Techniques for synthesizing the probes and primers described herein arewell-known in the art and include chemical syntheses and recombinantmethods. Such techniques are described in Sambrook et al., “MolecularCloning: A Laboratory Manual”, 3rd Edition, Cold Spring HarborLaboratory Press, (2001), incorporated herein by reference. Primers andprobes can also be made commercially (e.g., CytoMol, Sunnyvale, Calif.or Integrated DNA Technologies, Skokie, Ill.). Techniques for purifyingor isolating the probes and primers described herein are well-known inthe art. Such techniques are described in Sambrook et al., “MolecularCloning: A Laboratory Manual”, 3rd Edition, Cold Spring HarborLaboratory Press, (2001), incorporated herein by reference. The primersand probes described herein can be analyzed by techniques known in theart, such as restriction enzyme analysis or sequencing, to determine ifthe sequence of the primers and probes is correct.

In various embodiments of the methods and compositions described herein,the probes and primers can be labeled, such as with fluorescentcompounds, radioactive isotopes, antigens, biotin-avidin, colorimetriccompounds, or other labeling agents known to those of skill in the art,to allow detection and quantification of amplified DNA, such as byReal-Time PCR. In illustrative embodiments, the labels may include6-carboxyfluorescein (FAM™), TET™ (tetrachloro-6-carboxyfluorescein),JOE™ (2,7,-dimethoxy-4,5-dichloro-6-carboxyfluorescein), VIC™, HEX(hexachloro-6-carboxyfluorescein), TAMRA™(6-carboxy-N,N,N′,N′-tetramethylrhodamine), BHQ™, SYBR® Green, Alexa350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,Fluorescein, Oregon Green 488, Oregon Green 500, Oregon Green 514,Pacific Blue, REG, Rhodamine Green, Rhodamine Red, ROX, and/or TexasRed.

Specificity of the probes and primers described herein was demonstratedby testing hybridization of the probe and primers sets against 23different mold organisms (10 species of Aspergillus, 10 species ofPenicillium, 2 species of Stachybotyrous, and 1 specie of Fusarium).There were no cross-over reactions or cross-over detection noted for anyof the tested probe and primer sequences. Thus, the methods andcompositions (e.g., primers and probes) for amplification of fungal DNAare highly specific and avoid co-amplification of or do not co-amplifynon-specific nucleic acids.

In one illustrative embodiment, universal probes can be used to providea method for determining the presence of fungal DNA before conductingtarget-specific assays. In one embodiment, universal probes and primerscan be used to detect the presence of Aspergillus and Penicilliumspecies (see probes and primers for Fungal Universal Group 1 below). Inanother embodiment, universal probes and primers can be used to detectthe presence of Stachybotrys and Fusarium species (see probes andprimers for Fungal Universal Group 2 below). In these embodiments, theprobes and primers can be homologous for all targets of interest relatedto Aspergillus, Penicillium, Stachybotrys, and Fusarium species.

Fungal Universal Group 1 (SEQ ID NO: 73) UP1: 5′-cctcggatcaggtagggatac(SEQ ID NO: 74) UF1: 5′-atgcctgtccgagcgtcatt (SEQ ID NO: 75)UR1: 5′-ttcctccgcttattgatatg  Fungal Universal Group 2 (SEQ ID NO: 76)Up2: 5′-acggatctcttggctctggcatc (SEQ ID NO: 77)F2: 5′-gcggagggatcattaccgag (SEQ ID NO: 78)UR2: 5′-ttcactgaattctgcaattcac

In another illustrative embodiment, a method is provided of identifyinga mycotoxin in patient tissue or body fluid. The method comprises thesteps of extracting and recovering the mycotoxin from the patient tissueor body fluid, contacting the mycotoxin with an antibody directedagainst the mycotoxin, and identifying the myocotoxin.

Illustratively, patient (e.g., human or animal) tissue is received in1.) a 10% formalin fluid or 2.) in a paraffin block in which the tissuehas been fixed in formalin. In one embodiment for mycotoxin detectionand quantitation, the tissue can then be processed by variousdehydration steps and finally embedded in paraffin. In this embodiment,the tissue can then be cut in 3-5 micron samples. In an illustrativeembodiment, approximately 25-35 mg of tissue can then be processed asdescribed in Examples 2-6 for mycotoxin extraction. Illustratively, bodyfluids can be prepared as described in Examples 1 and 3-6 or by othermethods known in the art. In another illustrative embodiment, patientbody fluids can be tested for the presence of mycotoxins.Illustratively, any antigen associated with a fungus or with a mycotoxincan be detected.

The methods and compositions for detection and quantification ofmycotoxins are very specific and sensitive (e.g., sensitivity of 1.0ng/ml for aflatoxins, 0.2 ng/ml for Tricothecenes, and 2 ng/ml forochratoxins). Specificity of mycotoxins was tested in each group(Tricothecenes, Aflatoxins, Ochratoxins) by testing known samples ofmycotoxins (obtained from Trilogy Laboratories, Washington, Missouri,and from Sigma, St. Louis, Mo.) in each mycotoxin test. There were nocross-over reactions or cross-over detection of mycotoxins between thegroups. Thus, the methods and compositions for detection andquantification of mycotoxins are also very specific and sensitive. Themethods and compositions described in Examples 2-6 utilizeantibody-based identification of mycotoxins. In illustrativeembodiments, Enzyme-Linked Immunosorbant Assay (ELISA), affinitychromatography, or a Luminex®-based assay can be used to detectmycotoxins produced by toxic molds. Illustratively, the mycotoxins canbe aflatoxins, ochratoxins, or Tricothecenes (e.g., Verrucarins A, B andJ, Roridin A, E, H, and L-2, Satratoxins F, G, and H, Verrucarol,isosatratoxin F, G, and H, and T-2).

Illustrative of antibody-based assays that can be used to identify andsubsequently quantitate mycotoxins, or fungal or mycotoxin antigens, arethe Tricothecene kit (Envirologix, Inc., Portland, Me.), the AflaTest®(VICAM, Inc.), and the OchraTest™ (VICAM Inc., Watertown, Mass.).

Another exemplary detection method for multiple mycotoxins in patientsamples that have been exposed to fungal targets belonging to, forexample, Aspergillus, Penicillium, Stachybotrys, and Fusarium species isthe Luminex® format (Luminex, Austin, Tex., see Examples 9 and 11-13).In one aspect of the invention, the Luminex® assay utilizes microspheres(beads) that are dyed with fluorochromes and that are coupled toantigens to detect antibodies, in patient body fluids or tissues, tomycotoxins, mycotoxin antigens, or other fungal antigens. In anotherembodiment, the microspheres are coupled to antibodies to detect, inpatient body fluids or tissues, mycotoxins, mycotoxin antigens, or otherfungal antigens. In this illustrative embodiment, the antibodies coupledto the microspheres can be polyclonal or monoclonal antibodies, butmonoclonal antibodies are typically used. In another illustrativeembodiment, the beads can be coupled to DNA probes to detect DNAspecific to fungal species. Exemplary methods involving the Luminex®assay are described in more detail in Examples 9 and 11-13.

In the embodiment where mycotoxins are identified and quantitated,control samples of the body fluid or tissue to be analyzed can beobtained from patients with no documented history of exposure to moldsor mycotoxins. For example, negative control samples can be obtainedfrom autopsy specimens in which the patient had no exposure tomycotoxins or molds (e.g., victims of motor vehicle accidents, coronaryartery disease, or myocardial infarction). For positive controls, forexample, samples of negative tissue and/or body fluids can be spikedwith known positive amounts of mycotoxins or spores prior to evaluationto generate a calibration curve (see Examples 4-6). Illustrativecalibrators for the mycotoxins can be obtained from producers of theTricothecene kit (Envirologix, Inc., Portland, Me.) and producers of theAflaTest® and OchraTest™ kits (VICAM Inc., Watertown, Mass.), or fromTrilogy Laboratories (Washington, Mo.).

In another embodiment, a method is provided of determining if a patientis at risk for or has developed a disease state related to a fungalinfection. The method comprises the steps of extracting and recovering amycotoxin (i.e., a mycotoxin or a mycotoxin antigen) from a tissue orbody fluid of the patient, contacting the mycotoxin (i.e., a mycotoxinor a mycotoxin antigen) with an antibody directed against the toxin,identifying the mycotoxin (i.e. , a mycotoxin or a mycotoxin antigen),and determining if the patient is at risk for or has developed thedisease state related to the fungal infection. In another embodiment, amethod is provided of determining if a patient is at risk for or hasdeveloped a disease state related to a fungal infection. The methodcomprises the steps of extracting and recovering DNA of a specificfungal species from a tissue or body fluid of the patient, amplifyingthe DNA, hybridizing a probe to the DNA to specifically identify thefungal species, and specifically identifying the fungal species.

In any embodiment involving “determining if the patient has developedthe disease state related to the fungal infection,” this phrase means“diagnosing the patient with a fungal infection.”

The method embodiments described in the preceding paragraph providemethods of diagnosing fungal infections. Patients in need of diagnosisof a fungal infection can include cancer patients, post-operativepatients, transplant patients, patients undergoing chemotherapy,immunosuppressed patients, and the like. These patients may experiencesymptoms of fungal infections including sinusitis, allergic reactions,headaches, and skin rashes. Patients in need of diagnosis may includehumans or animals.

In one embodiment, for diagnosing fungal infections, kits are provided.The kits are useful for identifying, detecting, or quantitating fungalDNA or mycotoxins in a patient tissue or body fluid. In the embodimentwhere the kit is used to identify fungal DNA, the kit can contain theprobes and/or primers described herein, components to extract andisolate fungal DNA, and components for DNA amplification, such as a heatstable DNA polymerase (e.g., Taq polymerase or Vent polymerase),buffers, MgCl₂, H₂O, and the like. In the embodiment where the kit isused to identify mycotoxins (i.e., a mycotoxin or a mycotoxin antigen),the kit can contain components to extract and isolate the mycotoxin(i.e., a mycotoxin or a mycotoxin antigen), antibody affinity matrices,ELISA plates, Luminex® beads, polyclonal or monoclonal antibodies, colordevelopment reagents, buffers, and the like. In one embodiment, thereagents can remain in liquid form. In another embodiment, the reagentscan be lyophilized. In another illustrative embodiment, the kit can beused to detect other fungal antigens. The kits can also containinstructions for use.

In another embodiment, a kit comprising a purified nucleic acid with asequence selected from the group consisting of SEQ ID NO: 1 to SEQ IDNO: 89 or a complement of a sequence selected from the group consistingof of SEQ ID NO: 1 to SEQ ID NO: 89 is provided. The kit can comprisecomponents for the extraction and recovery of fungal DNA or a mycotoxinfrom the body fluid or tissue of a patient. The kit can further comprisecomponents for identification of the fungal DNA or the mycotoxin. Thecomponents for identification of the fungal DNA or the mycotoxin caninclude beads dyed with a fluorochrome and coupled to a probe for thefungal DNA or to antibodies to the mycotoxin or to the mycotoxin itselfor to a mycotoxin antigen.

A purified nucleic acid is also provided comprising a sequence of SEQ IDNO: 1 to SEQ ID NO: 89 or a sequence that hybridizes under highlystringent conditions to a sequence consisting of SEQ ID NO: 1 to SEQ IDNO: 89. A purified nucleic acid is also provided comprising a complementof a sequence of SEQ ID NO: 1 to SEQ ID NO: 89 or a sequence thathybridizes under highly stringent conditions to the complement of asequence consisting of SEQ ID NO: 1 to SEQ ID NO: 89. In accordance withthe invention “highly stringent conditions” means hybridization at 65°C. in 5×SSPE and 50% formamide, and washing at 65° C. in 0.5×SSPE.

A calibration reagent (or multiple calibration reagents) can also beincluded in the mycotoxin kit and “calibration reagent” means anystandard or reference material containing a known amount of themycotoxin (i.e., a mycotoxin or a mycotoxin antigen). The samplesuspected of containing the mycotoxin and the calibration reagent (ormultiple calibration reagents) are assayed under similar conditions. Themycotoxin concentration is then calculated by comparing the resultsobtained for the unknown sample with the results obtained for thecalibration reagent(s).

The following examples provide illustrative methods for carrying out thepractice of the present invention. As such, these examples are providedfor illustrative purposes only and are not intended to be limiting.

EXAMPLE 1 Samples and Sample Preparation

Human urine was received in 5-10 ml quantities as first in the morningvoided urines. Serums were received with the blood clot removed prior toreceipt and a minimum of 1 ml of serum was frozen or used. Nasalsecretions were obtained from hospital patients or out-patients. Fixedautopsy and surgical biopsy specimens were obtained from patients whohad a history of exposure to mycotoxins or fungi. These samples wereobtained from hospital pathology departments or coroners' offices.Tissue samples and body fluid samples were also obtained from patientswho had no exposure to mycotoxins or fungi and were sampled as anegative control group. Tissue specimens were cut using proceduresdescribed in Examples 2 and 7.

All specimens were placed into two groups. Group 1 comprised samplesfrom individuals with no reported symptoms or known fungi or mycotoxinexposure. These samples served as negative controls and n valuesdiffered in each group of specimens sampled. Group 2 comprised samplesfrom individuals with reported exposure to non-identified fungi orchemicals. Each test conducted had a different n value. Common symptomsof patients corresponding to group 2 samples included blurred vision,memory loss, fatigue, headache, nausea, loss of balance, cognitivedeficits, rhinitis, sinusitis, rashes, and allergies. A detailed historyand symptoms were provided to correspond to each patient sample.

Human urine was tested for the mycotoxins Aflatoxin B1, B2, G1, and G2,Ochratoxin A, and Tricothecenes. The studies described hereindemonstrated that higher levels of mycotoxins were detected in earlya.m. samples. Nasal secretions and washings were obtained by injectionof 3-5 ml of sterile saline in each nostril of a patient. The patientwas instructed to hold the saline in the nostrils for 30 seconds andthen blow the saline into a sterile container held close to the nose.The specimen(s) were then collected and placed in containers.

Negative control samples of mycotoxins were made by dilution techniquesfor Aflatoxin (Sigma), Ochratoxin (Sigma) and Roridin A (forTricothecenes). Samples of extracted and filtered human heart tissue,liver tissue, urine, and nasal secretions (including sputum) were spikedwith various levels of the above named toxins. Each time a sample wasevaluated, calibrators and negative and positive spiked tissues andfluids were also evaluated. Statistical analysis on all types of samplesfor mycotoxins were performed for sensitivity and specificity.

EXAMPLE 2 Preparation of Tissues for Mycotoxin Extraction

Preparation of tissues for myctotoxin extraction from formalin fixedtissue and paraffin-embedded tissue from humans or animals wasaccomplished using the following procedure.

Specimens

Tissue was received as either tissue fixed in a 10% formalin solution orin a paraffin-embedded tissue block. Tissue can be stored indefinitelyin either form. However, because of cross-linking of formalin andproteins which may give false negative readings for DNA, the tissue wasnot stored in formalin for greater than 6 months. A minimum of 25-35 mgof formalin-fixed tissue was required for mycotoxin extraction. Amaximum of 3 grams of formalin-fixed tissue can be used.

Materials

Phosphate Buffered Saline (PBS; 0.9%), acid-washed silica beads (Cat#G1277; obtained from Sigma-Aldrich), collection tubes (2 ml) screw cap,methanol (reagent grade, Sigma), and microcentrifuge tubes (2 ml) wereused.

Procedure

For silica beads, 0.3 g±0.01 g of silica bead beating glass was added toa 2 ml screw cap tube making sure that there were no glass beads in thecap or around the rim. The tubes containing the beads were sterilized inan autoclave on the dry cycle for 10 minutes. If a large amount oftissue was evaluated, the tissue was placed in a blender and blended inPBS until well emulsified in the PBS. The sample was then filtered usingsimple gravity filtration through Whatman #9 filter paper.

The samples were recorded and assigned numbers in a sample log. 25-35 mgof paraffin-embedded tissue was then weighed and placed in a 2.0 mlscrew cap tube. Methanol was added (1.0 ml reagent grade methanol) tothe tube with the 0.3 g of silica beads and the sample was vortexed for1 minute. The samples were bead beated on the bead beater for 1 minuteat the speed of 45. Then 500 μl of sample was removed and placed in 4.5ml of PBS taking care not to remove the paraffin from the sample tube.The sample could then be used for extraction or could be frozen at −20degrees centigrade to be used later in extraction and detection ofaflatoxins, ochratoxins, and tricothecenes (see Examples 3-6).

EXAMPLE 3 Preparation of Body Fluids for Mycotoxin Detection

Urine was received from a morning fresh first-voided specimen and storedat 1-6 degrees centrigrade in a glass container. A urine analysis wasconducted using a dipstick to measure pH, specific gravity, glucose,nitrates, ketones, and blood. The urine was examined for sediment andwas centrifuged at 2500 rpm for 5 minutes if sediment was present. Thesupernatant was saved in a glass container for mycotoxin testing(storing in plastic was avoided to avoid a decrease in the detectionlevel of tricothecenes). The urine was then used as indicated in Example4 for aflatoxin determination, Example 5 for ochratoxin determination,and Example 6 for tricothecene determination.

Nasal secretions and mucous samples as well as washes were observed formucous presence. If mucous was present, a solution of MUCOSOL™ (AlphaTec Systems, Inc. Vancouver, Wash.) was prepared and added in equalamounts of body fluid to MUCOSOL™ in the secretions containing mucous.The specimen was then allowed to incubate 30 minutes at roomtemperature. The specimen was then centrifuged and the supernatant wasremoved. The sediment was then re-suspended in 10 ml of PBS. Thespecimen was then treated like any other body fluid and subjected totests as described in Examples 4, 5, and 6. If testing for the presenceof fungal DNA was desired, the specimen was then subjected to the testsdescribed in Example 7.

Blood samples were obtained from the negative control group and exposedpatients. Specimens were allowed to clot (no anticoagulant added) andthen centrifuged for 10 minutes at 2000 rpm. Specimens were stored at1-6 degrees centigrade for 48 hours or were frozen at −20 degreescentigrade for an indefinite period of time. Blood samples wereextracted in a manner similar to that described by Garbis et al., Anal.Chem. 73:53589-64 (2001) and Hedman et al. Arch. Tieremahr. 50:13-24(1997). Serum samples were aliquoted in 200 μl amounts into sterile 1.5ml polystyrene microcentrifuge tubes. Immediately, 600 μl of highperformance HPLC grade acetonitrile (Fisher Scientific, Hampton, N.H.)was added. After 15 minutes, the samples were vortexed and centrifuged.The supernatants were transferred into clean 1.5 ml glass vials. Eachsample was evaporated under a gentle stream of dry nitrogen andre-suspended in 100 μl of pre-warmed sterile water. This was the finalworking solution for ELISA assays. Spinal fluid samples were analyzed asobtained from human patients. Samples were not processed beforeanalysis.

EXAMPLE 4 Detection of Aflatoxins in Body Fluids and Tissues

Aflatoxins are mycotoxins produced by the fungus Aspergillus. Samplesfrom body fluids and tissues were prepared as described in Examples 2and 3. Samples were then applied to an AflaTest® column (VICAM, L.P.,Watertown, Mass.) which contains specific monoclonal antibodies directedagainst Aflatoxins B1, B2, G1, and G2. The aflatoxins bind to theantibodies on the column. The column was then washed to remove theimmunoaffinity column of impurities. By passing an eluting solutionthrough the column, the aflatoxin was removed from the antibody. Theeluting solution was then read in a fluorometer. Aflatoxins B1, B2, G1,and G2 were detected using AflaTest® (VICAM, L.P., Watertown, Mass.)which is a well-known quantitative method for the detection ofaflatoxins in grains and foodstuffs. This assay permits the measurementof all of the major aflatoxins (including B1, B2, G1, G2 and M1) withoutthe use of toxic solvents (i.e., chloroform or methylene chloride).

In more detail, the procedure for body fluids and tissues was asfollows. Tissue samples were prepared from fixed and paraffin-embeddedtissues as described in Example 2 and body fluid samples were preparedas described in Example 3. One ml of the extracted tissue sample or bodyfluid was mixed with 10 ml of purified molecular grade water. The 10 mlof sample was then placed over an Aflatoxin® column (VICAM) and wasfiltered through the affinity column at a rate of 1-2 drops/second untilair came through the column. Ten ml of an 80:20 solution ofmethanol:water was then passed through the column at a rate of 2drops/second.

Another 10 ml of purified water was passed through the affinity columnat 2 drops/second. As an alternative procedure, ten ml of moleculargrade water can be passed through the column at a rate of 2drops/second. The affinity column was then eluted by passing 1.0 ml ofHPLC grade methanol through the column at a rate of 1-2 drops/second andcollecting all of the sample eluate (1 ml) in a glass cuvette. One ml ofAflaTest® Developer (described below) containing bromine was added tothe eluate in the cuvette. The specimen was then mixed well and thecuvette was placed in a calibrated fluorometer (Sequoia-Turner Model450). The aflatoxin concentration was read after 60 seconds.

Preparation of AFLATEST Developer Solution: Five ml of AflaTest®Developer concentrate solution (VICAM) was measured and placed in a 2ounce amber glass bottle of a 50 ml bottle dispenser for the developer(VICAM Cat. no. 20600). Fourty-five ml of purified water was added andmixed. The developer solution should be used within 8 hours afterpreparation. To ensure that the developer and water show nofluorescence, 1.0 ml of each was placed in the fluorometer and readafter 60 seconds. Results of the readings should be 0 ppb. Also,methanol blanks and purified water blanks should be read as 0 ppb. Theresults of the test were recorded in parts per billion(nanograms/mililiter).

Fluorometer Calibration: The Sequoia-Turner Fluorometer, Model 450, wascalibrated as defined by the manufacturer's guidelines (VICAM). Themachine was calibrated using standards supplied by VICAM (GreenStandard=−2, and Red Standard=22). Known standards of Aflatoxin B1, B2,G1, and G2 were used (Trilogy Laboratories, Washington, Mo.). StandardCalibrators were made at 52 ppb, 26 ppb, and 2.6 ppb.

Pump and Set Up: AflaTest® affinity chromatography was performed withthe AflaTest® affinity column attached to a pump stand. The stand has aglass syringe barrel that serves as a reservoir for the column. A largeplastic syringe with tubing and coupling provides air pressure tomanually push liquids through the column. An adjustable air pump (VICAMCat. no. 20650) can be attached to the pump tube instead of the largepump syringe barrel to operate without using hand pressure. Four and sixposition pump stands with aquarium pumps (VICAM cat. no. 21045) wereused when testing multiple specimens.

Aflatoxin Panel Results:

AFLATOXIN PANEL Specimen 0-0.5 ppb >0.5 ppb # of Tests CSF 2 0 2 NasalSecretions 19 4 23 Tissue Block 6 3 9 Urine 9 24 33 Other 3 0 3 Total:39 31 70

EXAMPLE 5 Detection of Ochratoxins in Body Fluids and Tissues

Ochratoxin A (OA) is detected using OchraTest® (VICAM, L.P., Watertown,Mass.) which is a well-known quantitative method for the detection ofOA. This assay permits the measurement of the major OA without the useof toxic solvents (i.e., chloroform or methylene chloride). Ochratoxinis a mycotoxin produced by the fungus Aspergillus ochraceous and also byseveral species of Penicillium fungi. To measure ochratoxin levels,samples from body fluids and tissues are prepared by subjecting samplesto the treatments described in Examples 2 and 3. The extracts are thenapplied to the OchraTest® column, which contains specific monoclonalantibodies for OA. The ochratoxin binds to the antibodies on the column.The column is then washed to remove impurities from the immunoaffinitycolumn. By passing an eluting solution through the column, theochratoxin is removed from the antibody. The eluting solution can thenbe measured in a fluorometer.

In more detail, the procedure for body fluids and tissues was asfollows. Tissue samples were prepared from fixed and paraffin-embeddedtissues as described in Example 2 and body fluid samples were preparedas described in Example 3. One ml of the extracted tissue sample,prepared sinus washes, or prepared urine was then mixed with 10 ml ofPBS. The 10 ml of sample was then placed over an OchraTest® column(VICAM) and filtered through the affinity column at a rate of 1-2drops/second until air came through the column. As an alternativeprocedure, 1.0 ml of sample can be mixed with 1.0 ml of 80:20 HPLC GradeMethanol: water solution. Then one (1) ml of the sample+80:20 HPLCMethanol: water solution can be added to 10 mls of PBS in eachimmunoaffinity column (OchraTest® column (VICAM)) and filtered throughthe affinity column at a rate of 1-2 drops/second until air came throughthe column. Ten ml of an 80:20 solution of methanol:water was thenpassed through the column at a rate of 2 drops/second. Ten ml of PBS wasthen passed through the affinity column at 2 drops/second. For thealternative procedure, these last two steps are not done. The affinitycolumn was then washed by passing 10 ml of 1× Mycotoxin Wash Buffer(OchraTest® VICAM) through the column at a rate of 1-2 drops/seconduntil air came through the column. The affinity column was then elutedby passing 1.5 ml of OchraTest® eluting solution through the column at arate of 1 drop/second and collecting all of the sample eluate (1.5 ml)in a glass cuvette. The sample was mixed well and the cuvette was placedimmediately into the calibrated fluorometer (Sequoia-Turner Model 450).The sample was read after 60 seconds. The results of the test arerecorded in parts per billion (nanograms/mililiter).

Fluorometer Calibration: The Sequoia-Turner Fluorometer, Model 450, wascalibrated as defined by the manufacturer's guidelines (VICAM). Themachine was calibrated using standards supplied by VICAM (GreenStandard=−1.5 and Red Standard=23; Mycotoxin Calibration Standards (1vial each of 3 levels) were used). Standards were prepared from controlspurchased from Trilogy Laboratories (Missouri). Standards were 50 ppb,25 ppb, and 2.5 ppb. The lower limit of detection was determined to be2.0 ppb using calibrators and known recovery rates of samples from theVICAM Immuno-affinity columns.

Pump and Set Up: OchraTest™ affinity chromatography was performed withthe OchraTest™ affinity column attached to a pump stand. The stand has aglass syringe barrel that serves as a reservoir for the column. A largeplastic syringe with tubing and coupling provides air pressure tomanually push liquids through the column. An adjustable air pump (VICAMCat. no. 20650) can be attached to the pump tube instead of the largepump syringe barrel to operate without using hand pressure. Four and sixposition pump stands with aquarium pumps (VICAM Cat. No. 21045) are usedwhen testing multiple specimens.

OCHRATOXIN PANEL Specimen 0-2.0 ppb >2.0 ppb # of Tests CSF 1 0 1 NasalSecretions 11 0 11 Tissue Block 6 1 7 Urine 18 4 22 Other 3 0 3 Total:39 5 44

EXAMPLE 6 Detection of Tricothecenes in Body Fluids and Tissues

This assay provides a procedure for the quantitative detection ofTrichothecenes including Roridin A, E, H and L-2, Satratoxin G and H,Isosatratoxin F, Verrucarin A and J, and Verrucarol in human tissue andhuman body fluids treated as described in Examples 2 and 3.

Tricothecenes were measured using a kit provided by Envirologic Inc,(ENVIROLOGIX QuantiTox Kit (EP-100) for Trichothecenes; Envirologic,Inc. Portland, Me.). The Envirologic kit is based on a competitiveEnzyme-Linked Immunosorbent Assay (ELISA). In the test, Trichothecenesin a sample compete with enzyme (horseradish peroxidase)-labeledsatratoxin for a limited number of antibody binding sites on the surfaceof the test wells. After a simple wash step, the outcome of thecompetition is visualized using a color development step. As with allcompetitive immunoassays, sample concentration is inversely proportionalto color development (i.e., darker color=lower concentration ofTrichothecenes in the sample and lighter color=higher concentration ofTrichothecenes in the sample).

All plate kit components were stored at 4-8° C. when not in use. The kitcomponents were never exposed to temperatures greater than 37° C. orless than 2° C. All reagents were allowed to reach ambient temperaturebefore use. Reagents or test well strips from one plate kit were notused with reagents or test well strips from a different plate kit.Substrate was not exposed to sunlight during pipetting or whileincubating samples in the test wells.

PBS Extraction/Dilution Buffer was prepared by making 1.0 liter of PBS,pH 7.4±0.05 in deionized water. PBS was stored refrigerated when notbeing used to prevent bacterial contamination. PBS was warmed to roomtemperature prior to use. Roridin A calibrators in PBSExtraction/Dilution Buffer were made daily in PBS using apositive-displacement pipet. At the end of the day, any unusedcalibrators were disposed of by adding to a 10% bleach solution. Thecalibrators were as follows:

-   -   18.0 ppb Roridin A calibrator=20 μl of 900 ppb stock solution        into 980 μl PBS in a glass tube.    -   2.0 ppb Roridin A calibrator=100 μl of 18 ppb calibrator into        800 μl PBS in a glass tube.    -   0.2 ppb Roridin A calibrator=10 μl of 18 ppb calibrator in 890        μl PBS in a glass tube.    -   Negative Calibrator: use an aliquot of the PBS stock.

Once all of the components had reached room temperature, the plate wasremoved from the pouch. All calibrators, sample extracts, and pipetteswere organized so that the samples and enzyme conjugates could be addedto the wells in 10 minutes or less. Fifty 50 μl of negative calibrator(NC), 50 μl of each Roridin A calibrator (C1-C3) and 50 μl of eachsample extract (S1-S8) were added rapidly to their respective wells.Fifty μl of Enzyme Conjugate (Satratoxin Enzyme Conjugate), was rapidlyadded to each well.

The contents of the wells were mixed thoroughly by moving the plateframe in a rapid circular motion on the bench top for 30-45 seconds. Thewells were covered with tape or parafilm and incubated at ambienttemperature for 45 minutes with or without shaking on an orbital shakerat 200 rpm. After incubation, the covering was removed and the contentsof the wells removed into a suitable container. The wells were floodedwith water and then shaken to empty the wells a total of five times. Theplate was then inverted several times on a paper towel to remove as muchwater as possible.

A 100 μl aliquot of substrate was then added to each well and thecontents thoroughly mixed as described in the preceding paragraph. Thewells were covered with new tape or parafilm and incubated for 15minutes at ambient temperature with or without an orbital shaker. Tostop the color development reaction, 100 μl of 1.0 N hydrochloric acidwas added to each well and the samples were mixed thoroughly. Thisturned the well contents yellow. The plate was read within 30 minutes ofthe addition of the HCl using a Multiskan MCC 341 Microplate Reader at awavelength of 450 nm.

New kit lots were tested in parallel with the kit lots currently in use.In order to test the new lot, one sample from the test batch wasprocessed twice in the same protocol using both the new kit lot and theold kit lot. If the results from the new lot differed from the currentlot, the lot test was repeated or the reagent was discarded.

A semi-log curve fit for the standard curve was used to plot the pointsof the calibrators. See below as example in the data reductionworksheet.

R{circumflex over ( )}2 = −0.9980 Slope = −0.3524 Intercept = 0.7950Samples were run in duplicate and were plotted to give results in partsper billion (ppb) or nanograms/ml (see FIGS. 1 and 2).

TRICOTHECENES PANEL Specimen 0-0.2 ppb >0.2 ppb # of Tests CSF 0 2 2Nasal Secretions 26 16 42 Tissue Block 5 8 13 Urine 5 104 109 Other 4 26 Total: 40 132 172

EXAMPLE 7 Detection of Fungal DNA

The target organisms for detection of fungal DNA using a PCR-basedapproach are shown in the following table. The table also indicates theprimer-probe set (number in parentheses in the table) used foramplification and detection. The primers and probes used foramplification and detection of fungal DNA are also shown below.

Targets:

Aspergillus Penicillium Stachybotyrous Fusarium Control (24) (15) (1)chartarum (14)solani (6) GEO amstelodami aurantiogriseum (7) flavus (4)chrsogenum (23) echinata (8) fumigatus (16) citrinum (9) nidulans (17)corylophilum (3) niger (18) crustosum (10) ochraceus (20) fellutanum(11) parasiticus (21) roquefortii (12) sydowii (22) simplisticum (13)ustus (5) verrucosum (2) versicolor (19) expansium

Primers and Probes: Target 1-S. chartarum (SEQ ID NO: 1) Probe 1 char:5′-ttgcttcggcgggaacgccccg (SEQ ID NO: 2) Primer F2:5′-gcggagggatcattaccgag (SEQ ID NO: 3) Primer R2:5′-atcgatgccagagccaagag Target 2-A. versicolor (SEQ ID NO: 4)Probe 2 vers: 5′-cggggagccctctcgggggc (SEQ ID NO: 5) Primer F1:5′-cgtaggtgaacctgcggaag (SEQ ID NO: 6) Primer R1:5′-atcgatgccggaaccaagag Target 3-A. niger (SEQ ID NO: 7) Probe 3 niger:5′-tgtctattgtacctgttgcttc (SEQ ID NO: 8) Primer F14:5′-cgtaggtgaacctgcggaag (SEQ ID NO: 9) Primer R1:5′-atcgatgccggaaccaagag Target 4-P. chrysogenum (SEQ ID NO: 10)Probe 4 chry: 5′-ctctgtctgaagattgtagtctgagt (SEQ ID NO: 11) Primer F1:5′-cgtaggtgaacctgcggaag (SEQ ID NO: 12) Primer R1:5′-atcgatgccggaaccaagag Target 5-P. verrucosum (SEQ ID NO: 13)Probe 5 verru: 5′-cccgcctttgctggccgcc (SEQ ID NO: 14) Primer F1:5′-cgtaggtgaacctgcggaag (SEQ ID NO: 15) Primer R1:5′-atcgatgccggaaccaagag Target 6-G. candidum (SEQ ID NO: 16)For Geo F1H: 5′-ggatctcttggttctcgtatc (SEQ ID NO: 17) Rev Geo R1H:5′-cttgatctgaggttgaatagtg (SEQ ID NO: 18) Probe 6 geo:5″-aacgcacattgcactttggggtatc Target 7-A. flavus (SEQ ID NO: 19)Probe 7 flay: 5′-cccgccattcatggccgccggg (SEQ ID NO: 20) Primer F1:5′-cgtaggtgaacctgcggaag (SEQ ID NO: 21) Primer R1:5′-atcgatgccggaaccaagag Target 8-A. fumigatus (SEQ ID NO: 22)Probe 8 fumi: 5′-aaagtatgcagtctgagttgattatc (SEQ ID NO: 23) Primer F1:5′-cgtaggtgaacctgcggaag (SEQ ID NO: 24) Primer R1:5′-atcgatgccggaaccaagag Target 9-A. nidulans (SEQ ID NO: 25)Probe 9 nid: 5′-cccagggggcgagccgccgg (SEQ ID NO: 26) Primer F1:5′-cgtaggtgaacctgcggaag (SEQ ID NO: 27) Primer R1:5′-atcgatgccggaaccaagag Target 10-A. ochraceus (SEQ ID NO: 28)Probe 10 ochr: 5′-acaccaacgtgaacactgtctgaag (SEQ ID NO: 29) Primer F1:5′-cgtaggtgaacctgcggaag (SEQ ID NO: 30) Primer R1:5′-atcgatgccggaaccaagag Target 11-A. paraciticus (SEQ ID NO: 31)Probe 11 para: 5′-cgggcccgccgtcatggccg (SEQ ID NO: 32) Primer F1:5′-cgtaggtgaacctgcggaag (SEQ ID NO: 33) Primer R1:5′-atcgatgccggaaccaagag Target 12-A. sydowii (SEQ ID NO: 34)Probe 12 syd: 5′-ccctcgggggcgagccgccg (SEQ ID NO: 35) Primer F1:5′-cgtaggtgaacctgcggaag (SEQ ID NO: 36) Primer R1:5′-atcgatgccggaaccaagag Target 13-A. ustus (SEQ ID NO: 37) Probe 13 ust:5′-ccacaccgaacctcttgttatagc (SEQ ID NO: 38) Primer F1:5′-cgtaggtgaacctgcggaag (SEQ ID NO: 39) Primer R1:5′-atcgatgccggaaccaagag Target 14-F. solani (SEQ ID NO: 40)Probe14salani: 5′-cgggaatagacggccccgtgaaac (SEQ ID NO: 41) Primer F2:5′-gcggagggatcattaccgag (SEQ ID NO: 42) Primer R2:5′-atcgatgccagagccaagag Target 15-P. aurantiogriseum (SEQ ID NO: 43)Probe 15 aurae: 5′-cccgcctttactggccgccgg (SEQ ID NO: 44) Primer F1:5′-cgtaggtgaacctgcggaag (SEQ ID NO: 45) Primer R1:5′-atcgatgccggaaccaagag Target 16-P. citrinum (SEQ ID NO: 46)Probe 16 citr: 5′-tgttgcctcggcgggccccgc (SEQ ID NO: 47) Primer F4:5′-ggaaggatcattaccgagtg (SEQ ID NO: 48) Primer R1:5′-atcgatgccggaaccaagag Target 17-P. corylophilum (SEQ ID NO: 49)Probe 17 corylo: 5′-ttattgtaccttgttgatcggcgg (SEQ ID NO: 50) Primer F1:5′-cgtaggtgaacctgcggaag (SEQ ID NO: 51) Primer R1:5′-atcgatgccggaaccaagag Target 18-P. crustosum (SEQ ID NO: 52)Probe 18 crust: 5′-cgatctccgggggacgggcc (SEQ ID NO: 53) Primer F7:5′-ctgtccgagcgtcattgctg (SEQ ID NO: 54) Primer R5: 5′-cgaggaccggacgcggtgTarget 19-P. expansum (SEQ ID NO: 55) Probe19expan:5′-agacacccccgaactctgcctgaa (SEQ ID NO: 56) Primer F1:5′-cgtaggtgaacctgcggaag (SEQ ID NO: 57) Primer R1:5′-atcgatgccggaaccaagag Target 20-P. fellutanum (SEQ ID NO: 58)Probe 20 fell: 5′-cccgcctgccaggccgccg (SEQ ID NO: 59) Primer F1:5′-cgtaggtgaacctgcggaag (SEQ ID NO: 60) Primer R1:5′-atcgatgccggaaccaagag Target 21-P. roquefortii (SEQ ID NO: 61)Probe 21 roque: 5′-cacccgtgtttatttaccttattgc (SEQ ID NO: 62) Primer F1:5′-cgtaggtgaacctgcggaag (SEQ ID NO: 63) Primer R1:5′-atcgatgccggaaccaagag Target 22-P. simplicissimum (SEQ ID NO: 64)Probe 22 simpl: 5′-cacccgtgtttatcgtaccttgttg (SEQ ID NO: 65) Primer F1:5′-cgtaggtgaacctgcggaag (SEQ ID NO: 66) Primer R1:5′-atcgatgccggaaccaagag Target 23-S. echinata (SEQ ID NO: 67)Probe 23 echin: 5′-ttgcttcggcgggagagccccg (SEQ ID NO: 68) Primer F2:5′-gcggagggatcattaccgag (SEQ ID NO: 69) Primer R2:5′-atcgatgccagagccaagag Target 24-E. amstelodami (SEQ ID NO: 70)Probe 24 amst: 5′-tgtctatctgtaccctgttgcttcg (SEQ ID NO: 71) Primer F1:5′-cgtaggtgaacctgcggaag (SEQ ID NO: 72) Primer R1:5′-atcgatgccggaaccaagag Fungal Universal Group 1 (SEQ ID NO: 73) UP1:5′-cctcggatcaggtagggatac (SEQ ID NO: 74) UF1: 5′-atgcctgtccgagcgtcatt(SEQ ID NO: 75) UR1: 5′-ttcctccgcttattgatatg Fungal Universal Group 2(SEQ ID NO: 76) Up2: 5′-acggatctcttggctctggcatc (SEQ ID NO: 77) F2:5′-gcggagggatcattaccgag (SEQ ID NO: 78) UR2: 5′-ttcactgaattctgcaattcac

Extraction Methods:

Bead Beater Tube Preparation:

-   -   1. 0.3 g±0.01 g of silica bead beating glass (Sigma-Aldrich Cat.        no G1277) was added to 2 ml screw cap tube avoiding glass beads        in the cap or around the rim.    -   2. The tubes containing the beads were sterilized in an        autoclave on the dry cycle for 10 minutes.    -   3. The tubes were removed from the autoclave (proceed to the        next step).

Solution Preparation:

-   -   4. Buffers ATL (from DNAeasy® Tissue Kit, Cat. no. 69506        (Quiagen, Stanford Valencia, Calif.)) and AL (from DNAeasy®        Tissue Kit, Cat. no. 69506) may form precipitates upon storage.        If a precipitate formed in either buffer, the buffer was        incubated at 55° C. until the precipitate fully dissolved.    -   5. Buffers AW1 and AW2 (from DNAeasy® Tissue Kit, Cat.        no. 69506) were supplied as concentrates. Before using for the        first time, the appropriate amounts of ethanol (96-100%) were        added to Buffers AW1 and AW2 as indicated on the bottles.    -   6. A 55° C. heat block and a 70° C. heat block were prepared for        use in the assay.

Preparation of the Spore Solution or Tissue:

-   -   7. If frozen material was used, it was equilibrated to room        temperature.    -   8. About 25.0 mg of paraffin-embedded tissue was weighed or 10.0        μl of spore solution was placed in a 2.0 ml screw cap tube.    -   9. 180.0 μl of ATL Buffer and 20.0 μl of Proteinase K was added        to each sample making sure that the lysate was not gelatinous.    -   10. 10.0 μl of the Geo Spore reference DNA was added to each        sample. (See Assay Specific Procedure for information regarding        internal and external controls)    -   11. All samples were bead beated on the Bead Beater for 1 minute        at the speed of 45.    -   12. Samples were incubated at 55° C. on a pre-warmed heat block        for 1 hour.

Extraction of Nucleic Acid:

-   -   15. The samples were removed from the heat block and vortexed 15        seconds.    -   16. 200 μl of Buffer AL was added and incubated at 70° C. for 10        minutes.    -   17. The tubes were removed from the 70° C. heat block and add        200 μl of ethanol    -   18. 200 μl of ethanol was added to each tube and vortexed.    -   19. The mixture underneath the layer of paraffin was pipetted        for each sample, making sure not to pipette the silica beads,        into the corresponding DNeasy® Mini Spin Column 2 ml collection        tube combo for that sample.    -   20. The columns were centrifuged in a microcentrifuge at 8000        RPM for 1 minute. The collection tube containing the flow        through was discarded.    -   21. Each spin column was placed in a new 2.0 ml collection tube.    -   22. 500.0 μl of Buffer AW1 was added to each column and        centrifuged at 8000 RPM for 1 minute. The collection tube        containing the flow through was discarded.    -   23. Each spin column was placed in a new 2.0 ml collection tube.    -   24. 500.0 μl of Buffer AW2 was added to each column and        centrifuged at 13,000 RPM for 5 minute.    -   25. The spin columns were removed carefully from the collection        tubes so as not to splash nozzles. The collection tube        containing the flow through was discarded.    -   26. The spin columns were placed in their corresponding 1.5 ml        elution tube.    -   27. 100.0 μl of Buffer AE (from DNAeasy® Tissue Kit, Cat.        no. 69506) was placed into each spin column and incubated for 3        minutes at room temperature.    -   28. The spin columns were centrifuged at 8000 RPM for 1 minute.        The spin columns were discarded and capped and the extracted        nucleic acid samples were stored at −20° C.

Real-Time PCR:

Preparation and Reaction Setup

1. Dilution of Probe Stocks

-   -   a. Resuspend the lyophilized probes in PCR grade water to a        final concentration of 100 μM.        -   (Example: If the synthesis yields 15.03 nMoles, add 150.3 of            PCR grade water to achieve 100 μM concentration)

2. Dilution of Primer Stocks

-   -   a. Resuspend the lyophilized primers in PCR grade water to a        final concentration of 100 μM.        -   (Example: If the synthesis yields 38.6 nMoles, add 386 μl of            PCR grade water to achieve 100 μM concentration)

3. Preparation of Primer/Probe Working Stock

-   -   a. See Appendix A for the working Stock setup for each target.

4. Reaction Setup

-   -   a. The reaction setup for one reaction is shown below. In some        cases the addition of MgCl₂ or varying concentrations of        primer/probe mix is required for PCR. (See Appendix A)

DNA  5.0 μl Primer/Probe Working Stock  3.5 μl (Final Concentration seeappendix A) OmniMix Beads  0.5 μl Beads (no volume contribution) PCRGrade Water 16.5 μl Total 25.0 μl

-   -   (Note that during reaction setup a master mix will be prepared        for multiple reactions using the Smart Cycler PCR Worksheet        (20.4007F). See following sections.)

5. Smart Cycler Cycling Parameters (Omni Fungal I)

-   -   a. Omni Fungal I is the primary program used for the fungal real        time assays and the run parameters for this program are outlined        below. Cases may occur where changes to this program may be        necessary for a specific target or specimen type. See        SmartCycler Operation (20.2008S) for further instruction on        programming and run optimization utilizing the Smart Cycler        software.        -   Step 1 (1 Cycle)        -   Hot Start: 95° C. for 120 seconds        -   Step 2 (45 cycles)        -   Denature: 95° C. for 5 seconds        -   Anneal: 60° C. for 45 seconds    -   See Example PCR Worksheet: (Note: sheet has been truncated to        show 3 target sets.)

II. Master Mix Setup

II. Master Mix Setup Set 1 Reagent Lot # Volume (uL) Reaction No. TotalAmount Target 1 H2O 4532 16.5 6 99.0 (3) A niger 2 P/P Working Stock040505 3.5 6 21.0 3 Omni Mix (Bead) 2456 0.5 6 3.0 4 MgCl2 NA 0.0 0 0.0Set 2 Reagent Lot # Volume (uL) Reaction No. Total Amount Target 1 H2O4532 16.5 6 99.0 (2) A Versicolor 2 P/P Working Stock 020705 3.5 6 21.03 Omni Mix (Bead) 2456 0.5 6 3.0 4 MgCl2 NA 0.0 0 0.0 Set 2 Reagent Lot# Volume (uL) Reaction No. Total Amount Target 1 H2O 4532 16.5 4 66.0(6) Geo 2 P/P Working Stock 020705 3.5 4 14.0 3 Omni Mix (Bead) 2456 0.54 2.0 4 MgCl2 NA 0.0 0 0.0 *Add MgCl₂ as needed per target subtractvolume used from water added to maintain a 20 μl reaction. Add 20 μl ofMaster Mix to each tube and then add 5.0 μl of template for a totalvolume of 25.0 μl.

Appendix A—Target Working Stock Recipes

Mix target working stocks for each target as described in the tableabove by combining the amounts noted for Primer 1, Primer 2, Probe,MgCl₂ (if needed), and water. Use 3.5 μl of this working stock for eachreaction performed.

Master Mix Preparation:

-   -   1. All steps were performed under sterile conditions.    -   2. After the water and beads had been pipetted into to the        individual tubes, the tubes were mixed until the beads (Cat no.        Omni 1-100N-050; Cepheid, Sunnyvale, Calif.) were completely        dissolved.    -   3. After the beads were dissolved, the appropriate primer/probe        working stock was pipetted into each master mix tube as        described in the PCR worksheet.    -   4. The solutions were mixed completely and the working stocks        returned to the −20° C. freezer.    -   5. Controls—        -   a. Internal Control—Every clinical sample processed was            inoculated with spores from the internal control target            Geometrica to show that a negative target result is a true            negative result and not related to the extraction of the            sample. The samples were processed through the extraction            protocol and amplified and detected utilizing primer and            probes specific for Geometrica.        -   b. Positive Control—A positive control for each target of            interest (Primer/Probe sets) was processed along with each            clinical sample in each real-time PCR run. This positive            control can be extracted from tissue or spore solutions but            must be lot checked prior to use. The positive control shows            that the primer/probe set for each target is not being            inhibited and shows that a negative result is a true            negative.        -   c. Negative Control—A negative control for each target of            interest (Primer/Probe sets) was processed along with each            clinical sample in each real-time PCR run. This negative            control can be extracted from tissue or water but must be            lot checked prior to use. The negative control shows that            the primer/probe set, water and extraction reagents for each            target is not contaminated with the target and shows that a            positive result is a true positive.

Addition of Target Nucleic acid:

-   -   1. 5.0 μl of the negative control, positive control and patient        samples was pipetted into the appropriate reaction tubes.    -   2. The reaction tubes were centrifuged using the Smart Cycler®        II modified centrifuge.    -   3. The tubes were returned to the cooling block and stored at        4° C. or the Smart Cycler Setup and Run was conducted.

Smart Cycler Setup and Run:

-   -   1. The Omni Fungal I protocol or the appropriate protocol was        selected for this real-time run.    -   2. For information regarding the operation of the Smart Cycler        see SmartCycler Operation (20.2008S) (Smart Cycler® II        Instrument; Cepheid, Sunnyvale, Calif.).

Data Analysis:

-   -   1. After the run is completed the results were analyzed by        reviewing each site in the results table. If a specific sample        tested was registered as positive by the software there was a        positive in the results column for that sample. There was also a        crossing point registered in the Ct column for that sample.    -   2. After reviewing the Results Table, the curves were reviewed        for each sample by selecting the “FAM” or “Log FAM” of the        “Views” menu.    -   3. With the graph selected, all samples that created a curve        were present on the screen. Each sample was reviewed        independently by clicking on the Site ID associated with the        sample of interest located just to the right of the graph.    -   4. A sample was analyzed as positive by the software if the        curve broke the baseline of 30 (default set in section above)        before the end of the 45 cycles and negative if it did not break        the baseline of 30 before the end of the 45 cycles.    -   5. Each sample was reviewed and then highlighted so that all        sample curves were present on the graph.

Results Interpretation:

-   -   1. Positive Result: A positive result is defined as any        amplification observed crossing a baseline fluorescence of ≧30        between cycles 1 and 40 of the real-time PCR run.    -   2. Negative Result: A negative result is defined as no        amplification observed crossing a baseline fluorescence of ≧30        between cycles 1 and 40 of the PCR run.    -   3. Equivocal Result: An equivocal result is defined as no        amplification observed crossing a baseline fluorescence of ≧30        between cycles 1 and 40, a control out of range or questions        regarding sample integrity.    -   4. Positive Control: A control that is positive for the target        being tested and shows that the assay will show a positive in        the presence of target spores and that there is not PCR        inhibition.    -   5. Negative Control: A control that is negative for the target        being tested and shows that the reagents or the sample were not        contaminated with the target prior to the testing of the sample.    -   6. Internal Control: A control used to show that the extraction        process is working fine for the purification of nucleic acid        from the clinical specimen and that a negative result is truly        negative and not due to an issue associated with the extraction.        (Note: the internal control must be positive for any sample to        be reported as negative for a target.)

See Table Below:

Reportable Crossing Positive Negative Internal Result Point ControlControl Control Positive ≧40 (+) (−) (+) Result Positive ≧40 (−) (−) (+)Result Positive ≧40 (+) (−) (−) Result Positive ≧40 (−) (−) (−) ResultNegative (−) (+) (−) (+) Result Negative (−) (+) (+) (+) Result Negative(−) (−) (+) (+) Result Un- reportable Crossing Positive NegativeInternal Result Point Control Control Control Positive ≧40 (+) (+) (+)Result Positive ≧40 (−) (+) (+) Result Positive ≧40 (+) (+) (−) ResultPositive ≧40 (−) (+) (−) Result Negative (−) (−) (−) (+) Result Negative(−) (+) (−) (−) Result Negative (−) (+) (+) (−) Result EquivocalCrossing Positive Negative Internal Result Point Control Control ControlCase by Case Case by Case Case by Case Case by Case Case by CaseIn other illustrative embodiments, results can be determined based on acycle range between cycles 1 and 45 of the PCR run or other usefulranges can be used.

DNA Results

Pos Results/ Neg Results/ Tissue tissue tissue Isolates ** Lung 6 1Aspergillus fellutanum (1) Asp. niger (2) P. chrysogenum (3) Liver 5 2Asp. flavus (2) Asp. niger (3) Pen. versicolour (2) Asp. fumigatus (1)Brain 4 2 Asp. niger (2) Asp. fumigatus (2) Asp. flavus (2) Skin 2 1Penicillium fellutanum (1) Pen. chrysogenum (1) Asp. ustus (1)Respirator^(##) 3 6 Asp. niger (2) Asp. flavus (2) Pen. versicolour (1)** some specimens revealed more than one isolate ^(##)includes nasalsecretions, inner ear fluids, and sputums

The test is 100% specific for the isolates tested for.

15 known negative (non-exposed) patients were tested in validation. Nopositive results with the probes in this patent were found.

Of the 29 suspected exposed patients tested, 29 samples of tissue weretested. Of those 29 tissues/fluids, 20 specimens gave results of one ormore organisms by DNA probing using RT-PCR.

EXAMPLE 8 Detection of Fungal DNA Using Universal Primers

The purpose of the universal fungal assays is to check clinical samplesfor the presence of fungal DNA prior to running multiple fungal targetspecific assays. Two universal assays were designed for this purpose.Fungal assay UP1 was designed to detect the presence of fungalAspergillus and Penicillium species. Fungal assay UP2 was designed todetect the presence of Stachybotrys and Fusarium species. Each assay iscomposed of two primers and one Taqman probe specific for fungal targetsof the species described above.

To identify sequence present in 1.) all Aspergillus and Penicilliumspecies, and in 2.) all Stachybotrys and Fusarium species a search wasperformed for public sequence and sequence was identified. A sequencingprimer (oligonucleotide˜20 bases long) was designed specific for each ofthe targets of interest, and was ordered from a vender. The primers andtarget DNAs were sequenced, and the accuracy confirmed. Primers andprobes were designed and were expected to be specific for all individualtargets in each species group as described below. To check forspecificity to the species groups described, the sequences of each assaywere put through a “Blast” search against known sequences from fungalgenomes. Initial results showed these sequences to be specific for thespecies as described above and not specific for clinically relevanttargets outside the species.

The following sequences were found to be homologous for all targets ofinterest related to Aspergillus and Penicillium:

Assay UP1

(SEQ ID NO: 74) UF1 atgcctgtccgagcgtcatt (Forward Primer)(SEQ ID NO: 75) UR1 ttcctccgcttattgatatg (Reverse Primer)(SEQ ID NO: 73) UP1 cctcggatcaggtagggatac (Taqman probe)

The following sequences were found to be homologous for all targets ofinterest related to Stachybotrys and Fusarium:

Assay UP2

(SEQ ID NO: 77) F2 gcggagggatcattaccgag (Forward Primer) (SEQ ID NO: 78)UR2 ttcactgaattctgcaattcac (Reverse Primer) (SEQ ID NO: 76Up2 acggatctcttggctctggcatc (Taqman probe)

EXAMPLE 9 Detection of Mycotoxins Using Luminex®

The purpose of this assay is to utilize the Luminex® platform to detectapproximately 3 toxin groups (tricothecenes, aflatoxins, andochratoxins) in patient samples that have been exposed to fungal targetsbelonging to Aspergillus, Penicillium, Stachybotrys, and Fusariumspecies. The Luminex® assay utilizes microspheres (beads) that arecoupled to antigens to detect antibodies against those specific antigensin a sample. Samples and coupled microspheres will be incubated inmicrotitration filter wells where antigen-antibody binding occurs. Afterincubation and washing, the appropriate detection antibody (e.g.,biotinylated antibody) will be introduced and incubated during whichantibody-antibody binding occurs. After incubation and washing, areporter conjugate will be added and incubated where the biotin-bindingreaction occurs.

In theory, each microsphere is color-coded into 100 different sets. Eachbead set can be coated with a reagent to capture and detect a specificanalyte from a sample. The Luminex® 100 has lasers that excite theinternal dyes that identify the microsphere and any reporter dyecaptured during the assay. During the run on the Luminex®, severalreadings will be made on each of the bead sets. Potentially, this willcreate a multiplexing capability of up to 100 unique assays with onesingle sample.

EXAMPLE 10 Detection of Fungal DNA and Mycotoxins in Human PatientsFindings for Patient #1 (Skin Biopsy)

-   Positive for P. chrysogenum and A. ustrus-   Positive for Trichothecenes (38 ppb)

Findings for Patient #2 (Endometriosis DX in Ovary and Uterus)

-   Positive for Trichothecenes (25 ppb in ovary pathology specimen)    Findings for Patient #3 (autopsy specimen)-   Positive for A. fellutanum (lung)-   Positive for A. flavus and A. niger (liver)-   Positive for A. niger (brain)

Findings for Patient #4 (Questionable Fungal Mass on Neck)

-   Positive for A. niger (left and right inner ear)-   Positive (left ear) for aflatoxin (9.5 ppb), ochratoxin (4.5 ppb),    and Trichothecenes (0.62 ppb)-   Positive (right ear) for aflatoxin (0.73 ppb) and Trichothecenes    (0.62 ppb)

Findings for Patient #5 (Pilocystic Astrocytoma, Brain Tumor)

-   6 year old female, Grade 1-2 Pilocystic astrocytoma, Brain-   RT-PCR: Negative for 23 probes-   Mycotoxins: aflatoxins (3.0 ppb), all other mycotoxins negative.

Findings for Patient #6 (Diagnosis: Pulmonary Fibrosis, Potential LungTransplant Patient)

-   38 year old male on oxygen. Previous lung biopsy: severe    Interstitial fibrosis (UIP).-   All bacterial and fungal cultures negative.-   RT-PCR: negative for 23 probes.-   Mycotoxins: aflatoxin (4 ppb), all other mycotoxin negative-   Treated with antifungal agent, and, after six weeks, he improved and    was taken off lung transplant emergent list. Oxygen use decreased    significantly.

Findings for Patient #7: (Headache and Seizures)

-   32 year old female with severe headaches and seizures. All    diagnostic tests negative. All cultures negative.-   RT-PCR: positive for Stachybotrys chartarum.

Findings for Patient #8 (Mouth Lesions for Two Years, No Resolution)

-   84 year old female with three mouth lesions, chief complaint: pain    and mouth feels like “cotton”.-   Biopsy: parakeratosis, ulceration, no malignant cells, chronic    inflammation, increased vascularity. Diagnosis: consistent with    history of aphthous stomatitis.-   RT-PCR: positive for Aspergillus sydowii.

Findings for Patient #9 (Chronic Headaches)

-   40 year old male with chronic headaches-   Pathology biopsy: Temporal dura and lobe: Perivascular inflammatory    cell infiltrate. No neoplasm identified. No organisms noted by stain    or culture or PCR for virus or bacteria.-   RT-PCR: positive for Aspergillus ustus and Emericella amstelodami.-   Mycotoxins: Negative    Findings for Patient #10 (Patient with Skin Lesions)-   56 year old male exposed to Stachybotrys, Aspergillus niger, and    Penicillium sp. (confirmed by Environmental evaluation).-   RT-PCR: Skin Biopsy: Positive for Stachybotrvs chartarum,    Aspergillus niger.-   Mycotoxins: Positive for Aflatoxin (4ppb); Negative for    tricothecenes.

EXAMPLE 11 Detection of Mycotoxins

The purpose of this assay is to utilize the Luminex® platform to detectapproximately 29 toxins in patient samples which include but are notlimited to blood, spinal fluid, urine, nasal secretions, sputum, andtissue that have been exposed to fungal targets belonging toAspergillus, Penicillium, Stachybotrys, and Fusarium species.

The Luminex® assay can, for example, utilize microspheres (beads) thatare coupled to monoclonal antibodies to detect mycotoxin antigens. Themonoclonal antibodies will be made or purchased for use in thisprocedure. The antigens are, for example, aflatoxin B1, B2, G1, and G2,Ochratoxin A, and a group of Trichothecenes. The group of Trichothecenesincludes Roridin A, E, H and L-2, Satratoxin G and H, Isosatratoxin F,Verrucarin A and J, and Verrucarol, and T-2. Samples and coupledmicrospheres will be incubated in microtitration filter wells whereantigen-antibody binding occurs. After incubation and washing, theappropriate detection antibody (e.g., biotinylated antibody) will beintroduced and incubated during which antigen-antibody binding occurs.After incubation and washing, a reporter conjugate will be added andincubated where the biotin-binding reaction occurs.

Each microsphere is color-coded into, for example, 100 different sets.Each bead set can be coated with a reagent to capture and detect aspecific analyte from a sample. The Luminex® 100 has lasers that excitethe internal dyes that identify the microsphere and any reporter dyecaptured during the assay. During the run on the Luminex®, severalreadings will be made on each of the bead sets. Potentially, this assayswill create a multiplexing capability of up to 100 unique assays with asingle sample.

EXAMPLE 12 Luminex Assay Using DNA Probes Bound to Beads

Microspheres (Luminex Corporation, Austin; Tex.) are 5.6 μm in diameterand are comprised of polystyrene, divinyl benzene, and methacrylic acidwith surface carhoxylate functionality for covalent attachment ofbiomolecules. The microspheres-are internally dyed with red,infrared-emitting fluorochromes. Spectral addresses were created byadjusting the concentrations of each fluorochrome with each bead set.When the microsphere sets were analyzed with the Luminex 100 instrument(Luminex), each bead set was identified and classified by a distinctfluorescence signature pattern.

Coupling: Two microsphere sets were used for covalent coupling of A.versicolor and F. solani probes to carboxyl functional groups on themicrosphere surfaces. A concentration of 5.0×10⁶ microspheres were addedto separate 1.5 ml microcentrifuge tubes and pelleted bymicrocentrifugation and the supernatant was aspirated. The microspherepellet was resuspended and vortexed in 50 μl of 0.1M2-(N-morpholino)-ethanesulfonic acid (MES), pH 4.5, 0.2 nanomole of each5′ amino-modified capture oligonucleotide or probe (A. versicolor:5′-CGGGGAGCCCTCTCGGGGGC-3′ (SEQ ID NO: 4) and F. solani:5′-CGGGAATAGACGGCCCCGTGAAAC-3′ (SEQ ID NO: 40)) and 25 μg of1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) (10mg/ml in dH₂O), and the mixture was incubated for 30 minutes at roomtemperature in the dark. The microsphere mixture was vortexed and 25 μgof EDC was added again and mixed, and the mixture was incubated for 30min at room temperature in the dark. One milliliter of 0.02% Tween-20(0.02% in dH₂O) was added to the coupled microspheres andmicrocentrifuged. The supernatant was removed and the pellet wasresuspended in 100 μl of 1× Tris EDTA, pH 8.0 (TE) (diluted in dH₂O), byvortex and sonication (see FIG. 3).

Assay: A working mixture of the microspheres was prepared at aconcentration of 150 microspheres per microliter in 1.5× TMAC (5M TMAC,20% Sarkosyl, 1M Tris-HCL, 0.5M EDTA, H₂O), vortexed and sonicated.Thirty-three microliters of each microsphere mixture was added to thewells of a 96-well V-bottom PCR plate and 17 μl TE, pH 8.0, were addedto the background wells. To each appropriate well 5′ biotinylatedcomplementary oligonucleotides (A. versicolor:5′-GCCCCCGAGAGGGCTCCCCG-3′ (SEQ ID NO: 90) and F. solani:5′-GTTTCACGGGGCCGTCTATTCCCG-3′ (SEQ ID NO: 91)) (5 to 200 femtomoles)and TE, pH 8.0, to a total well volume of 50 μl were added. The reactionwells were mixed, covered and incubated at 96° C. for 3 min to denatureany secondary structure. The reactions were incubated for 15 min at 52°C. and 25 μl of a 10 μg/ml dilution of streptavidin-R-phycoerythrin in1× TMAC (1.5× TMAC diluted in dH₂O) was added, mixed and incubated for 5min at 52° C. Fifty microliters from each well was analyzed at 52° C.with a minimum bead count set at 100 events.

Averaged DataType: Median Total Sample (fmol) A. versicolor Events 0 96215 5 1143 213 10 1344 215 20 1420 223 50 2513 205 100 4467 224 200 5206202

Averaged DataType: Median Total Sample (fmol) F. solani Events 0 66 2245 1470 213 10 1682 215 20 1740 205 50 1884 221 100 1974 210 200 1978 223

copied from output DataType: Median A. Location Sample versicolor TotalEvents 1 0 73 220 2 5 1358 221 3 10 1461 227 4 20 1639 230 5 50 2512 2016 100 3886 229 7 200 5279 201 8 0 56 200 9 0 118 209 10 5 928 205 11 101227 202 12 20 1201 215 13 50 2514 209 14 100 5047 218 15 200 5133 20316 0 104 226 DataType: Median Location Sample F. solani Total Events 170 99 210 18 5 1582 214 19 10 1636 204 20 20 1800 200 21 50 1887 225 22100 1932 212 23 200 1960 219 24 0 136 223 25 0 32 237 26 5 1359 211 2710 1728 226 28 20 1681 210 29 50 1882 216 30 100 2017 207 31 200 1997227 32 0 94 204

DNA Coupling Raw Data SN LX10001089024 Session022107couplingconfirmarion.RTL Operator Samples 32 Min Events 0 ResultsDataType: Median Total Location Sample Aver Fsol Events Notes 1 0 73 2202 5 1358 221 3 10 1461 227 4 20 1639 230 5 50 2512 201 6 100 3886 229 7200 5279 201 8 0 56 200 9 0 118 209 10 5 928 205 11 10 1227 202 12 201201 215 13 50 2514 209 14 100 5047 218 15 200 5133 203 16 0 104 226 170 99 210 18 5 1582 214 19 10 1636 204 20 20 1800 200 21 50 1887 225 22100 1932 212 23 200 1960 219 24 0 136 223 25 0 32 237 26 5 1359 211 2710 1728 226 28 20 1681 210 29 50 1882 216 30 100 2017 207 31 200 1997227 32 0 94 204 DataType: Count Total Location Sample Aver Fsol Events 10 100 120 220 2 5 121 100 221 3 10 100 127 227 4 20 130 100 230 5 50 100101 201 6 100 129 100 229 7 200 100 101 201 8 0 100 100 200 9 0 109 100209 10 5 105 100 205 11 10 102 100 202 12 20 115 100 215 13 50 109 100209 14 100 118 100 218 15 200 103 100 203 16 0 100 126 226 17 0 110 100210 18 5 114 100 214 19 10 104 100 204 20 20 100 100 200 21 50 125 100225 22 100 112 100 212 23 200 119 100 219 24 0 100 123 223 25 0 100 137237 26 5 100 111 211 27 10 126 100 226 28 20 100 110 210 29 50 116 100216 30 100 100 107 207 31 200 100 127 227 32 0 104 100 204

The data indicate that DNA probes can be bound to beads and can be usedto detect the presence of fungal DNA in samples.

EXAMPLE 13 Luminex Indirect Assay Using Antigen Bound to Beads

Microspheres (Luminex Corporation, Austin, Tex.) are 5.6 μm in diameterand are comprised of polystyrene, divinyl benzene, and methacrylic acidwith surface carboxylate functionality for covalent attachment ofbiomolecules. The microspheres are internally dyed with red,infrared-emitting fluorochromes. Spectral addresses were created byadjusting the concentrations each fluorochrome with each bead set. Whenthe microsphere sets were analyzed with the Luminex 100 instrument(Luminex®), each bead set was identified and classified by a distinctfluorescence signature pattern.

Coupling: Two microsphere sets were used for covalent coupling ofmycotoxin antigens. Aflatoxin (B1, B2, G1, G2) and Deoxynivalenol (DON)were used and required chemical modifications of carboxyl functionalgroups on the microsphere surfaces to amine groups due to the absence ofan alpha-amino N-terminal group on either antigen. Adipic aciddihydrazide (ADH) was used to modify the carboxyl functional groups onthe microsphere surfaces to provide an NH₂ group for coupling to eachantigen. A concentration of 1.25×10⁷ microspheres were added to separate1.5 ml microcentrifuge tubes and were washed by adding 500 μl of 100 mM2-(N-morpholino)-ethanesulfonic acid (MES), pH 6.0, microcentrifuged at10,000×g for 1 min at room temperature and the supernatant wasaspirated. The microsphere pellet was resuspended and vortexed in 1 mlof ADH and 200 μl of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) (200 mg/ml in 100 mM MES, pH 6.0) and the tubes wererotated for 1 h at room temperature in the dark. The microspheres werethen washed twice with 1 ml of 100 mM MES, pH 4.5, bymicrocentrifugation as described above, and the supernatants werediscarded. Five hundred microliters of each antigen (500 ppb Aflatoxinand 125 ppb DON) were added to each pellet of ADH-modified microspheresand the mixture was incubated at room temperature for 4 h with rotation.To remove any noncovalently bound mycotoxin, the microspheres werewashed twice by centrifugation with PBS-Tween and were blocked withPBS-Tween (see FIG. 4).

Immunoassay: In order to test the coupling efficiency of each antigen,Aflatoxin (B1, B2, G1, G2) and Deoxynivalenol (DON), to each microsphereset, an indirect immunoassay must be performed. For the competitiveassay, each mycotoxin antibody will be titrated in a two-fold dilutionand incubated in micotiter wells of a 96-well plate containing mycotoxinantigen-coupled microspheres. The MFI signal should increase as theantibody is diluted to reach optimal antibody dilution required for thecompetitive immunoassay. Two blanks will be identified with a “0” whichindicates the absence of mycotoxin antibody and a signal of less than 50MFI (Median Fluorescent Signal). The increasing signal indicating thelabeled mycotoxin antibody binding to the antigen-coupled microspheresand the low signal in the blank wells, where no labeled mycotoxinantibody is present, prove that the mycotoxin antigens were effectivelycoupled to the microsphere sets and can be detected by the appropriatemycotoxin antibodies.

Fifty microliters of each microsphere mixture were added to the wells ofa 1.2 μm-pore-size filter membrane microtiter plate. Fifty microlitersof PBS-1% BSA were added to each background well. Sheep Anti-Aflatoxinand Sheep Anti-DON antibodies were purified by ammonium sulfateprecipitation and desalted utilizing desalting spin columns and werelabeled with biotin using sulfosuccinimidyl-6-[biotin-amido]hexanoate.Each mycotoxin antibody was diluted in PBS-1% BSA in 2-fold dilutionsand 50 μl of each dilution were added to the standard wells. The wellswere mixed by pipetting and the samples were incubated, with shaking, atroom temperature in the dark for 1 h. The wells were aspirated using avacuum manifold filtration system and were washed twice with 100 μl ofPBS-1% PBS-BSA. The microspheres were resuspended in 50 μl PBS-1% BSAand 50 μl of 4 μg/ml Streptavidin-R-phycoerythyrin was added to eachwell, mixed and incubated, with shaking, at room temperature in the darkfor 30 min. The wells were washed twice with 100 μl PBS-1% BSA and thesamples were resuspended in 100 μl PBS-1% BSA. Seventy-five microlitersof each sample were analyzed with a minimum bead count set at 100events.

-   Data: Mycotoxin DON and Aflatoxin were each coupled to two sets of    Luminex microspheres. Biotinylated Sheep Anti-DON and Sheep    Anti-Aflatoxin were titrated in a two-fold dilution to show a dose    response.

Aflatoxin

Median DataType: Antibody Location Dose Aflatoxin Total Events 1 0 10100 2 0 11 100 3 Neat 106 100 4 1:2 218 100 5 1:4 152 100 6 1:8 868 1007  1:16 2442 100 8  1:32 4166 100

DON

Median DataType: Antibody Location Dose DON Total Events 1 0 4 100 2 0 4100 3 Neat 8 100 4 1:2 13 100 5 1:4 89 100 6 1:8 149 100 7  1:16 1451100 8  1:32 2162 100

Protein Raw Data for Microsphere Binding for detection of Mycotoxins.Program Luminex 100 IS Build 2.2 Date 3/3/2007 3:16:21 PM SN LX10001089024 Session 030307Afla.modbead.biotinAB Operator Samples 8 MinEvents 0 Results DataType: Median Total Location Sample Aflatoxin Events1 Blank 10 100 2 Blank 10.5 100 3 Neat Alfa 105.5 100 4 1:02 217.5 100 51:02 152 100 6 1:02 868 100 7 1:02 2442 100 8 1:02 4165.5 100 DataType:Count Total Location Sample Aflatoxin Events 1 Blank 100 100 2 Blank 100100 3 Neat Alfa 100 100 4 1:02 100 100 5 1:02 100 100 6 1:02 100 100 71:02 100 100 8 1:02 100 100 Program Luminex 100 IS Build 2.2 Date3/3/2007 3:13:49 PM SN LX 10001089024 Session030307DONmodbead.antibiotin Operator Samples 8 Min Events 0 ResultsDataType: Median Total Location Sample DON Events 1 Blank 3.5 100 2Blank 3.5 100 3 Neat Don 7.5 100 4 1:02 12.5 100 5 1:02 89 100 6 1:02148.5 100 7 1:02 1450.5 100 8 1:02 2161.5 100 DataType: Count TotalLocation Sample DON Events 1 Blank 100 100 2 Blank 100 100 3 Neat Don100 100 4 1:02 100 100 5 1:02 100 100 6 1:02 100 100 7 1:02 100 100 81:02 100 100The data indicate that mycotoxins or mycotoxin antigens can be bound tobeads and can be used to detect in a competive assay the presence ofantibodies to mycotoxins in samples.

EXAMPLE 14 Detection of Fungal DNA and Mycotoxins in Animals Findingsfor Dog (Necropsy, High Exposure to Environmental Molds):

Liver and Lung:

-   -   Mycotoxins: tricothecenes 0.96 ppb    -   RT-PCR: Penicillium chrysogenium and Aspergillus niger

Findings for Horse (Urine):

-   -   Tricothecenes: 5.32 ppb

Findings for Cat (Necropsy):

-   -   Aflatoxin: 4 ppb

1-58. (canceled)
 59. A method of identifying a specific fungal speciesin patient tissue or body fluid, the method comprising the steps of:extracting and recovering DNA of the fungal species from the patienttissue or body fluid; amplifying the DNA; hybridizing a probe to the DNAto specifically identify the fungal species, wherein the probe has asequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO:19, and SEQ ID NO: 22; and specifically identifying the fungal species.60. The method of claim 59 wherein the amplifying step is performed withprimers that hybridize to the DNA.
 61. The method of claim 59 whereinthe DNA is amplified using PCR.
 62. The method of claim 61 wherein thePCR is real-time PCR.
 63. The method of claim 59 wherein the probe isfluorescently labeled.
 64. The method of claim 62 wherein the probe isfluorescently labeled.
 65. The method of claim 60 wherein the probe hasthe sequence of SEQ ID NO:
 7. 66. The method of claim 60 wherein theprobe has the sequence of SEQ ID NO:
 19. 67. The method of claim 60wherein the probe has the sequence of SEQ ID NO:
 22. 68. The method ofclaim 60 wherein the amplified sequence is internal transcribed spacerregions of nuclear ribosomal DNA.
 69. The method of claim 59 wherein theprobe is bound to a bead dyed with a fluorochrome.
 70. A method ofdetermining if a patient is at risk for or has developed a disease staterelated to a fungal infection, the method comprising the steps of:extracting and recovering DNA of a specific fungal species from a tissueor body fluid of the patient; amplifying the DNA; hybridizing a probe tothe DNA to specifically identify the fungal species, wherein the probehas a sequence selected from the group consisting of SEQ ID NO: 7, SEQID NO: 19, and SEQ ID NO: 22; and specifically identifying the fungalspecies.
 71. The method of claim 70 further comprising the step ofdeveloping an effective treatment regimen for the patient.
 72. Themethod of claim 70 wherein the probe is bound to a bead dyed with afluorochrome.
 73. The method of claim 70 wherein the probe has thesequence of SEQ ID NO:
 7. 74. The method of claim 70 wherein the probehas the sequence of SEQ ID NO:
 19. 75. The method of claim 70 whereinthe probe has the sequence of SEQ ID NO:
 22. 76. A kit comprising apurified nucleic acid with a sequence selected from the group consistingof SEQ ID NO: 7, SEQ ID NO: 19, and SEQ ID NO: 22 or with a complementof a sequence selected from the group consisting of SEQ ID NO: 7, SEQ IDNO: 19, and SEQ ID NO:
 22. 77. The kit of claim 76, wherein the purifiednucleic acid has the sequence of SEQ ID NO:
 7. 78. The kit of claim 76,wherein the purified nucleic acid has the sequence of SEQ ID NO:
 19. 79.The kit of claim 76, wherein the purified nucleic acid has the sequenceof SEQ ID NO: 22.